Compositions and methods for modulating muscle cell and tissue contractility

ABSTRACT

The present invention relates to compositions and methods comprising one or more domains of urokinase-type plasminogen activator (uPA) in an amount effective to modulate one or more of the contractility and angiogenic activity of a mammalian muscle or endothelial cell or tissue for use in the treatment of a disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The one or more domains of uPA can be present in the inventive compositions and methods either as part of the full uPA molecule in either single chain or two chain form (scuPA or tcuPA), or as an isolated polypeptide, or a fragment of the uPA molecule (e.g., the amino terminal fragment “ATF”), or a deletion mutant of the uPA molecule. The inventive methods comprise administering to a mammal afflicted with such a disease or condition the inventive composition, and modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue, thereby treating the disease or condition. Kits for treating such diseases are also included.

This application claims benefit of U.S. Provisional Application No.60/212,874 filed Jun. 20, 2000.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH AND DEVELOPMENT

This invention was supported in part by U.S. Government funds (NIH GrantNo. HL 60169), and the U.S. Government may therefore have certain rightsin the invention.

BACKGROUND OF THE INVENTION

Urokinase-type plasminogen activator (uPA) is a serine protease whichhas been implicated in various biological processes, includingfibrinolysis, (Carmeliet et al., 1994, Nature 369:419-424; Carmeliet etal., 1996, Haemostasis 26:132-153; Pinsky et al., 1998, J. Clin. Invest.102:919-928; Bugge et al., 1996, Proc. Natl. Acad. Sci. USA93:5899-5904) angiogenesis, (Odekon et al., 1992, J. Cell, Physiol.150:258-263; Bacharach et al., 1992, Proc. Natl. Acad. Sci. USA89:10686; Pepper et al., 1990, J. Cell Biol. 111:743-755; Goldberg etal., eds., 1997, In: Regulation of Angiogenesis Birkhauser Verlag,Basel, pp. 391-411) neointima and aneurysm formation, (Clowes et al.,1990, Circ. Res. 67:61-67; Carmeliet et al., 1997, Circ. Res.81:829-839; Shireman et al., 1997, J. Vasc. Surg. 25:157-164; Noda-Heinyet al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:37-43; Lijnen et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18:1035-1045) chemotaxis,(Pedersen et al., 1996, Br. J. Haematol. 95:45-51) and wound healing(Carmeliet et al., 1998, J. Cell Biol. 140:233-245).

Certain of these activities may require the proteolytic activity of uPA,(Kirschheimer et al., 1987, Fed. Am. Soc. of Exper. Biol. and Med.Journal 1:125-128; DePetro et al., 1994, Exp. Cell Res. 213:186-194)whereas others involve intracellular signaling through the urokinasereceptor (uPAR) (Pedersen et al., 1996, Br. J. Haematol. 95:45-51;Degryse et al., 1999, Blood 94:649-662) or additional, as yet undefinedreceptors (Carmeliet et al., 1998, J. Cell Biol. 140:233-245; Kanse etal., 1997, Arteriosclerosis, Thromb., and Vas. Biol. 17:2848-2854;Koopman et al., 1998, J. Biol. Chem. 273:33267-33272; Rabbani et al.,1992, J. Biol. Chem. 267:14151-14156).

Urokinase is synthesized as a single chain molecule (scuPA) whichexhibits little or no intrinsic enzymatic activity (Urano et al., 1988,Arch. Biochem. Biophys. 264:222-230; Gurewich et al., 1987, Semin.Thromb. Hemost. 13:146-151; Husain, 1991, Biochemistry 30:5707-5805).scuPA is a multi-domain protein composed of a C-terminal protease domainand an amino-terminal fragment (ATF). The ATF is composed of twodomains: a growth factor domain (GFD) which binds to uPAR, and a kringledomain (uPA kringle), the function of which has heretofore been unknown.scuPA can be cleaved by plasmin at the Lys¹⁵⁸-Ile¹⁵⁹ position togenerate an enzymatically active, disulfide-linked two-chain urokinasemolecule (tcuPA). Between the ATF and the protease domain is a regiondesignated the connecting peptide (corresponding to amino acids136-158).

It is hypothesized that the natural enzyme uPA is normally used in thehuman body to dissolve clots and to facilitate cell migration. AlthoughuPA is known in the art as a useful therapeutic molecule for thetreatment of diseases and disorders having as a symptom thereof abnormalclotting in critical blood vessels, there remains a need in the art forcompositions and methods which are useful for the treatment of suchdiseases as well as for the treatment of diseases and disorders havingas symptoms abnormally high or abnormally low muscle cell contractilityor undesirable angiogenic activity. Such diseases and conditions includethe following: cardiovascular diseases and conditions such ashypotension, hypertension and atherosclerosis; thrombotic conditionssuch as stroke, heart attack and post angioplasty stenting; angiogenicdisorders; respiratory diseases and conditions such as pulmonaryfibrosis and asthma; diseases and disorders related to tumor cellinvasion, angiogenesis and metastasis; wound healing and clottingdisorders and reproductive disorders such as premature uterinecontraction and impotence. The present invention meets these needs.

BRIEF SUMMARY OF THE INVENTION

The invention includes a composition comprising the urokinase-typeplasminogen activator (uPA) kringle in an amount effective to modulateone or more of the contractility and the angiogenic activity of amammalian muscle or endothelial cell or tissue. The uPA kringle sharesat least about 75% homology with a polypeptide having the amino acidsequence corresponding to SEQ ID NO:1.

In one embodiment, the composition further comprises one or more domainsof uPA selected from the group consisting of the growth factor domain,the connecting peptide and the protease domain.

The invention also includes a composition comprising the growth factordomain of uPA in an amount effective to modulate the contractility of amammalian muscle cell or tissue. The growth factor domain shares atleast about 75% homology with a polypeptide having the amino acidsequence corresponding to SEQ ID NO:2.

In one embodiment, the composition further comprises one or more domainsof uPA selected from the group consisting of the uPA kringle, theconnecting peptide and the protease domain.

The invention also includes a composition comprising a polypeptide, thepolypeptide (LMW-uPA) comprising the connecting peptide and proteasedomains of uPA in an amount effective to inhibit the contractility of amammalian muscle cell or tissue. The polypeptide shares at least about75% homology with a polypeptide having the amino acid sequencecorresponding to SEQ ID NO:5.

In one aspect, the cell is in a mammal.

In another aspect, the muscle cell is selected from the group consistingof a smooth muscle cell, a striated muscle cell and a cardiac musclecell, and the muscle tissue is selected from the group consisting of asmooth muscle tissue, a striated muscle tissue and a cardiac muscletissue.

In one embodiment, the composition further comprises an inducingcompound in an amount effective to mediate the contraction of amammalian muscle cell or tissue. The inducing compound is selected fromthe group consisting of phenylepherine, epinepherine, acetylcholine andendothelin.

In another aspect, the composition comprises two chain urokinase(tcuPA). The tcuPA shares at least about 75% homology with a polypeptidehaving the amino acid sequence corresponding to SEQ ID NO:3.

In one embodiment, the composition comprises single chain urokinase(scuPA). The scuPA shares at least about 75% homology with a polypeptidehaving the amino acid sequence corresponding to SEQ ID NO:3.

In another embodiment, the composition comprises the amino terminalfragment (ATF) of uPA. The ATF shares at least about 75% homology with apolypeptide having the amino acid sequence corresponding to SEQ ID NO:4.

In one aspect, the uPA kringle is an isolated kringle.

In another aspect, the growth factor domain is an isolated growth factordomain.

In yet another aspect, the ATF is an isolated ATF.

In one embodiment, modulating the contractility of the muscle cell ortissue comprises enhancing or disinhibiting the contractility of themuscle cell or tissue.

In another embodiment, modulating the contractility of the muscle cellor tissue comprises enhancing or disinhibiting the contractility of themuscle cell or tissue.

In one aspect, the cell or tissue is a vascular smooth muscle orendothelial cell or tissue, and the uPA kringle is present in an amounteffective to modulate the angiogenic activity of the cell or tissue.

In another aspect, the cell or tissue is a vascular smooth muscle cellor tissue or a vascular endothelial cell or tissue.

In one embodiment, modulating the contractility of the muscle cell ortissue comprises inhibiting the contractility of the muscle cell ortissue.

In another embodiment, modulating the contractility of the muscle cellor tissue comprises inhibiting the contractility of the muscle cell ortissue.

In one aspect, the cell or tissue is a bronchial smooth muscle cell ortissue.

In one embodiment, the composition comprises the deletion mutantpolypeptide scuPA^(Δ136-143) in an amount effective to enhance ordisinhibit the contractility of a mammalian muscle cell or tissue,wherein the scuPA^(Δ136-143) shares at least about 75% homology with apolypeptide having the amino acid sequence corresponding to SEQ ID NO:6.

In another embodiment, the composition comprises a deletion mutantpolypeptide selected from the group consisting of Δkringle-scuPA andΔkringle-tcuPA in an amount effective to proteolytically activateplasminogen and to inhibit the contractility of a mammalian muscle cellor tissue, wherein the Δkringle-scuPA and the Δkringle-tcuPA each shareat least about 75% homology with a polypeptide having the amino acidsequence corresponding to SEQ ID NO:7.

In a further embodiment, the composition comprises a polypeptide, thepolypeptide comprising the amino terminal fragment (ATF) and theconnecting peptide of uPA, wherein the polypeptide shares at least about75% homology with a polypeptide having the amino acid sequencecorresponding to SEQ ID NO:8.

In yet a further embodiment, the composition comprises a polypeptide,the polypeptide comprising the uPA kringle and the connecting peptide,wherein the polypeptide shares at least about 75% homology with apolypeptide having the amino acid sequence corresponding to SEQ ID NO:9.

In one aspect, the composition is in the form of a pharmaceuticalcomposition.

The invention also includes a composition comprising one or morepolypeptides, each of the polypeptides having an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8and SEQ ID NO:9. The one or more polypeptides are present in an amounteffective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.

The invention also includes a composition comprising an isolated nucleicacid. The isolated nucleic acid has a nucleotide sequence which sharesat least about 75% homology with a nucleotide sequence selected from thegroup consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ IDNO:18. The isolated nucleic acid is present in the composition in anamount effective to transform a mammalian muscle or endothelial cell toprovide transgene expression of a polypeptide at a level of expressioneffective to modulate one or more of the contractility and angiogenicactivity of the muscle or endothelial cell after transfection with theisolated nucleic acid.

The invention includes a method of treating a mammal afflicted with adisease or condition having as a symptom thereof one or more of abnormalmuscle cell or tissue contractility and abnormal muscle or endothelialcell or tissue angiogenic activity. The method comprises a)administering to the mammal a composition comprising the uPA kringle inan amount effective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue,wherein the uPA kringle shares at least about 75% homology with apolypeptide having the amino acid sequence corresponding to SEQ ID NO:1;and b) modulating one or more of the contractility and the angiogenicactivity of the muscle or endothelial cell or tissue having one or moreof abnormal contractility and abnormal angiogenic activity, whereby thedisease or condition in the mammal is treated.

In one embodiment, the uPA kringle is a part of a polypeptide whichshares at least about 75% homology with a polypeptide selected from thegroup consisting of SEQ ID NO:3 (tcuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6(scuPA^(Δ136-143)), SEQ ID NO:8 and SEQ ID NO:9.

In one aspect, the composition further comprises one or more of anagonist of the uPA kringle, an agonist of a binding protein of the uPAkringle, an antagonist of the uPA growth factor domain, an antagonist ofthe connecting peptide, an antagonist of a binding protein of the uPAgrowth factor domain, and an antagonist of a binding protein of theconnecting peptide.

In another aspect, the disease or condition is selected from the groupconsisting of hypotension, hypertension, atherosclerosis, stroke, heartattack, microvascular occlusions, thrombotic microangiopathies,surgically induced thrombotic disorders, angiogenic disorders, pulmonaryfibrosis, asthma, tumor cell invasion, tumor cell angiogenesis, tumorcell metastasis, glaucoma diabetic retinopathy, a wound healing orclotting disorder, a uterine contraction disorder and male impotence.

The invention also includes a method for treating a mammal afflictedwith a disease or condition having as a symptom thereof one or more ofabnormal muscle cell or tissue contractility and abnormal muscle orendothelial cell or tissue angiogenic activity. The method comprises a)administering to the mammal a composition comprising the uPA growthfactor domain in an amount effective to modulate one or more of thecontractility and the angiogenic activity of a mammalian muscle orendothelial cell or tissue, wherein the uPA growth factor domain sharesat least about 75% homology with a polypeptide having the amino acidsequence corresponding to SEQ ID NO:2; and b) modulating one or more ofthe contractility and the angiogenic activity of the muscle orendothelial cell or tissue having one or more of abnormal contractilityand abnormal angiogenic activity, whereby the disease or condition inthe mammal is treated.

In one aspect, the composition comprises the uPA growth factor domain aspart of a polypeptide which shares at least about 75% homology with apolypeptide having the amino acid sequence selected from the groupconsisting of SEQ ID NO:3 (scuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6(scuPA^(Δ136-143)), SEQ ID NO:7 (Δkringle-scuPA or Δkringle-tcuPA) andSEQ ID NO:8.

In one embodiment, the composition further comprises one or more of anagonist of the uPA growth factor domain, an agonist of the connectingpeptide, an agonist of a binding protein of the growth factor domain, anagonist of a binding protein of the connecting peptide, an antagonist ofthe uPA kringle, and an antagonist of a binding protein of the uPAkringle.

In one aspect, the composition is administered to the mammal in anamount effective to inhibit the contractility of a mammalian smoothmuscle cell or tissue.

In another aspect, the smooth muscle cell or tissue is a vascular smoothmuscle cell or tissue, and the disease or condition treated ishypertension.

In another embodiment, the disease or condition is a respiratory diseaseor condition selected from the group consisting of asthma, adultrespiratory distress syndrome, primary pulmonary hypertension,microvascular thrombotic occlusion and a disorder associated withchronic intrapulmonary fibrin formation.

In one aspect, the uPA kringle is present in an amount effective toinhibit the contractility of a bronchial smooth muscle cell or tissueand is a part of a polypeptide selected from the group consisting of anisolated kringle, ATF, tcuPA, scuPA^(Δ136-143), SEQ ID NO:8 and SEQ IDNO:9.

In one embodiment, the disease or condition in the mammal sought to betreated has as a symptom thereof abnormally low vascular smooth musclecell or tissue contractility.

In another aspect, the uPA kringle is present in an amount effective toenhance or disinhibit the contractility of a vascular smooth muscle cellor tissue and is a part of a polypeptide selected from the groupconsisting of an isolated kringle, ATF, tcuPA, scuPA^(Δ136-143), SEQ IDNO:8 and SEQ ID NO:9.

In one embodiment, the disease or condition has as a symptom thereofabnormally high vascular smooth muscle cell or tissue contractility.

In one aspect, the uPA growth factor domain is present in thecomposition in an amount effective to inhibit the contractility of avascular smooth muscle cell or tissue, and is present in the compositionas a part of a polypeptide selected from the group consisting of anisolated growth factor domain, scuPA, Δkringle-scuPA and Δkringle-tcuPA.

The invention also includes a method of identifying a compound which isan agonist or antagonist of one or more of the uPA kringle or a bindingprotein thereof, the uPA growth factor domain or a binding proteinthereof, and the connecting peptide or a binding protein thereof, uponthe contractility or angiogenic activity of a mammalian muscle orendothelial cell or tissue. The method comprises a) providing to a firstcell and an otherwise identical second cell a composition comprising apolypeptide, the polypeptide comprising one or more of the uPA kringle,the uPA growth factor domain and the connecting peptide, wherein thepolypeptide is present in the composition in an amount effective tomodulate the contractility or angiogenic activity of a mammalian muscleor endothelial cell or tissue; b) providing to the first cell a testcompound; c) assessing the contractility or the angiogenic activity ofthe first cell and the second cell prior to and after administering thecomposition and the test compound to the first cell, and prior to andafter administering the composition to the second cell; and d) comparingthe contractility or angiogenic activity of the first cell with thecontractility or angiogenic activity of the second cell prior to andafter administration of the composition and the test compound. When theeffect of the composition upon contractility or angiogenic activity inthe first cell is either increased or decreased relative to the effectof the composition upon contractility or angiogenic activity in thesecond cell, a compound is identified which is an agonist or antagonistof one or more of the uPA kringle or a binding protein thereof, the uPAgrowth factor domain or a binding protein thereof, and the connectingpeptide or a binding protein thereof, upon the contractility orangiogenic activity of a mammalian muscle or endothelial cell or tissue.

The invention also includes a method of treating a disease or conditionin a mammal having as a symptom thereof one or more of abnormal musclecell or tissue contractility and abnormal angiogenic activity. Themethod comprises a) administering to the mammal an amount suspected tobe effective for modulating the contractility or angiogenic activity ofa mammalian muscle or endothelial cell or tissue of an agonist orantagonist of one or more of the uPA kringle or a binding proteinthereof, the uPA growth factor domain or a binding protein thereof, andthe connecting peptide or a binding protein thereof; b) providing theagonist or antagonist to a muscle or endothelial cell or tissue in themammal having abnormal contractility or abnormal angiogenic activity, orto a tissue or fluid which is contiguous therewith; and c) modulatingthe effect of one or more of the uPA kringle or a binding proteinthereof, the uPA growth factor domain or a binding protein thereof, andthe connecting peptide or a binding protein thereof, upon the muscle orendothelial cell or tissue having abnormal contractility or abnormalangiogenic activity, whereby a disease or condition in the mammal havingabnormal muscle cell or tissue contractility or abnormal angiogenicactivity as a symptom thereof is treated.

In one embodiment, the disease or condition treated is the vasculardisease hypertension.

In another embodiment, the agonist or antagonist is one or more of anantagonist to the uPA kringle, an antagonist to a binding protein of theuPA kringle, an agonist of the uPA growth factor domain, an agonist of abinding protein of the uPA growth factor domain, an agonist of theconnecting peptide, and an agonist of a binding protein of theconnecting peptide.

In one aspect, the disease or condition treated is selected from thegroup consisting of asthma, adult respiratory distress syndrome, primarypulmonary hypertension, microvascular thrombotic occlusion and adisorder associated with chronic intrapulmonary fibrin formation.

In another aspect, the agonist or antagonist is one or more of anagonist to the uPA kringle and an agonist to a binding protein of theuPA kringle.

The invention also includes a method of identifying whether a testprotein is a binding protein of one or more of the uPA kringle, the uPAgrowth factor domain and the connecting peptide. The method comprises a)assessing the contractility modulating effect or the angiogenic activitymodulating effect of one or more of the uPA kringle, the uPA growthfactor receptor and the connecting peptide upon a first cell or tissue,wherein the first cell or tissue comprises the test protein or iscontiguous with a tissue or fluid of a mammal which comprises the testprotein; b) assessing the contractility modulating effect or theangiogenic activity modulating effect of one or more of the uPA kringle,the uPA growth factor receptor and the connecting peptide upon a second,otherwise identical cell or tissue which does not comprise the testprotein and which is not contiguous with a tissue or fluid whichcomprises the test protein; and c) comparing the contractilitymodulating effect or the angiogenic activity modulating effect in thefirst cell or tissue with the contractility modulating effect or theangiogenic activity modulating effect in the second cell or tissue. Ifthe contractility modulating effect or the angiogenic activitymodulating effect of one or more of the uPA kringle, the uPA growthfactor receptor and the connecting peptide is greater in the first cellor tissue relative to the second cell or tissue, then the test proteinis a binding protein of one or more of the uPA kringle, the uPA growthfactor receptor and the connecting peptide.

Also included in the invention is a method of identifying a functionalelement of one or more of the uPA kringle, the uPA growth factor domainand the connecting peptide, the functional element participating in themodulation of contractility or angiogenic activity of a mammalian muscleor endothelial cell or tissue. The method comprises a) preparing one ormore mutant polypeptides which lack a portion of the amino acid sequenceof one or more of the uPA kringle, the uPA growth factor domain and theconnecting peptide; b) assessing the ability of each of the mutantpolypeptides to modulate the contractility or angiogenic activity of amammalian muscle or endothelial cell or tissue once provided to the cellor tissue, or to a tissue or fluid which is contiguous with the cell ortissue; c) identifying, from b) a mutant polypeptide which is not ableto modulate the contractility or angiogenic activity of a mammalianmuscle or endothelial cell or tissue; and d) determining from c) and a)the corresponding deleted portion of the amino acid sequence of one ormore of the uPA kringle, the uPA growth factor domain and the connectingpeptide which participates in the modulation of muscle or endothelialcell or tissue contractility or angiogenic activity, whereby afunctional element of one or more of the uPA kringle, the uPA growthfactor domain and the connecting peptide is identified.

The invention also includes a method of treating a vascular disease orcondition in a mammal having as a symptom thereof abnormally high fibrinclot formation. The method comprises a) administering to the mammal acomposition comprising one or more of Δkringle-scuPA, Δkringle-tcuPA, anantagonist of the uPA kringle and an antagonist of a binding protein ofthe uPA kringle in an amount effective to inhibit the contractility of amammalian vascular smooth muscle cell or tissue, wherein theΔkringle-scuPA and Δkringle-tcuPA share at least about 75% homology withthe polypeptide corresponding to SEQ ID NO:7; b) providing thecomposition to an affected vascular smooth muscle cell or tissue of thecardiovascular system of the mammal which has or is prone to excessivefibrin clot formation, or to a tissue or fluid which is contiguoustherewith; and c) vasodilating the affected vascular smooth muscle cellor tissue by inhibiting the contractility of the affected vascularsmooth muscle cell or tissue, thereby promoting both fibrin clot lysisand vasodilation in the affected area of the vasculature of the mammal,thereby treating the vascular disease or condition.

The invention also includes a kit for treating a disease or condition ina mammal, the disease or condition having as a symptom thereof one ormore of abnormal muscle cell or tissue contractility and abnormalangiogenic activity. The kit comprises a) a composition comprising apolypeptide, the polypeptide comprising one or more of the uPA kringle,the uPA growth factor domain, and the connecting peptide in an amounteffective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue;and b) an instructional material.

In one aspect, the kit further comprises a sterile solvent suitable fordissolving or suspending the composition prior to administering thecomposition to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1, comprising FIGS. 1A-1R, is a listing of the amino acid andnucleotide sequences SEQ ID NOs:1-18.

FIG. 1A is the amino acid sequence of the uPA kringle (SEQ ID NO:1).

FIG. 1B is the amino acid sequence of the uPA growth factor domain (SEQID NO:2).

FIG. 1C is the amino acid sequence of scuPA and tcuPA (SEQ ID NO:3).

FIG. 1D is the amino acid sequence of ATF (SEQ ID NO:4).

FIG. 1E is the amino acid sequence of LMW-uPA (SEQ ID NO:5).

FIG. 1F is the amino acid sequence of scuPA^(Δ136-143) (SEQ ID NO:6).

FIG. 1G is the amino acid sequence of Δkringle-scuPA and Δkringle-tcuPA(SEQ ID NO:7).

FIG. 1H is the amino acid sequence of ATF+the connecting peptide (SEQ IDNO:8).

FIG. 1I is the amino acid sequence of the uPA kringle+the connectingpeptide (SEQ ID NO:9).

FIG. 1J is the nucleotide sequence SEQ ID NO:10.

FIG. 1K is the nucleotide sequence SEQ ID NO:11.

FIG. 1L is the nucleotide sequence SEQ ID NO:12.

FIG. 1M is the nucleotide sequence SEQ ID NO:13.

FIG. 1N is the nucleotide sequence SEQ ID NO:14.

FIG. 1O is the nucleotide sequence SEQ ID NO:15.

FIG. 1P is the nucleotide sequence SEQ ID NO:16.

FIG. 1Q is the nucleotide sequence SEQ ID NO:17.

FIG. 1R is the nucleotide sequence SEQ ID NO:18.

FIG. 2 is a graph depicting the effect of uPA upon the contraction ofrat aortic rings in an accumulation curve. The contraction of rat aorticrings was induced by phenylepherine (PE) at the indicted concentrationsin the absence (♦) or presence of 10 nM scuPA (▴) or tcuPA (▪). In thisand in each of the remaining figures, the molar concentration of PE isdisplayed in logarithmic units. The mean±SD of 6 experiments is shown.

FIG. 3 is a graph depicting the stimulation of PE-inducedvasoconstriction by the amino-terminal fragment of uPA (ATF). Aorticrings were contracted by sequential addition of increasingconcentrations of PE in K—H buffer (▪) or K—H buffer supplemented with10 nM ATF (♦) or 10 nM ATF+10 nM suPAR (). The mean±standard deviationof three experiments is shown.

FIG. 4 is a graph depicting the stimulation of PE-inducedvasoconstriction by the uPA kringle. Aortic rings were contracted bysequential addition of increasing concentrations of PE in K—H buffer(—X—) or K—H buffer supplemented with increasing concentrations of uPAkringle: 1 nM (); 10 nM (—♦—); 50 nM (—▪—); 100 nM (). The mean±standarddeviation of three experiments is shown.

FIG. 5, comprising FIGS. 5A and 5B is a pair of graphs depicting theeffect of uPA^(Δ136-143) and Δkringle-uPA on PE-inducedvasoconstriction. FIG. 5A depicts the results after aortic rings wereincubated with 10 nM tcuPA^(Δ136-143) (—▪—), 10 nM scUPA^(Δ136-143)(—▴—) or PBS control (—♦—) and then with increasing concentrations of PEas described in the legend to FIG. 2. The mean±standard deviation ofthree experiments is shown. FIG. 5B depicts the results after aorticrings were incubated with 10 nM Δkringle-scuPA (), 10 nM Δkringle-tcuPA(—▴—) or PBS control (—♦—) and then with increasing concentrations of PEas described in the legend to FIG. 2. The mean i standard deviation ofthree experiments is shown.

FIG. 6, comprising FIGS. 6A, 6B and 6C, is a series of graphs depictingbinding of scuPA^(Δ136-143) to suPAR measured by surface plasmonresonance. The binding of wild type scuPA (FIG. 6A) and scUPA^(Δ136-143)(FIG. 6B) to suPAR are shown. Urokinase (12.5 nM to 0.2 nM) was thenadded in two fold increments (FIG. 6C). An experiment representative ofthree experiments so performed is shown.

FIG. 7 is a schematic depicting the amino acid sequence of the full uPAprotein.

FIG. 8 is a graph depicting the effect of the uPA kringle upon theAC-induced contraction of isolated rat tracheal rings. Isolated rattracheal rings were either pre-incubated with media containing 10nanomolar (—▴—) or 100 nanomolar (—♦—) uPA kringle or with control media(—▪—) lacking the uPA kringle, and then with varying concentrations ofacetylcholine (AC). The tension in the rings upon AC-induced contractionwas measured and is shown as a percent of the contraction of thecontrol. The mean±standard deviation of three independent experiments isshown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the finding that several domains of theurokinase-type plasminogen activator (uPA) are effective for modulatingthe contractility or modulating the angiogenic activity of a mammalianmuscle or endothelial cell or tissue. The invention includescompositions and methods for using these uPA domains advantageously tomodulate the contractility and/or the angiogenic activity of a mammalianmuscle or endothelial cell or tissue in the treatment of a disease orcondition having abnormal muscle cell or tissue contractility orabnormal muscle or endothelial cell or tissue angiogenic activity as asymptom thereof, or wherein inducing relaxation of a muscle cell ortissue or modulating angiogenesis would be efficacious to the mammal.

The invention also includes methods for identifying and using bindingproteins as well as agonists and antagonists of these domains of uPA inthe treatment of a disease or condition having abnormal muscle cell ortissue contractility or abnormal angiogenic activity as a symptomthereof In addition, agonists or antagonists to the binding proteins areused in the methods of the invention for the treatment of a disease orcondition having abnormal muscle cell or tissue contractility orabnormal angiogenic activity as a symptom thereof.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “isolated polypeptide” refers to a polypeptidesegment or fragment which has been separated from sequences which flankit in a naturally occurring state, e.g., a polypeptide fragment whichhas been removed from the sequences which are normally adjacent to thefragment, e.g., the sequences adjacent to the fragment in a protein inwhich it naturally occurs. The term also applies to a polypeptide whichhas been substantially purified from other components which naturallyaccompany the polypeptide, e.g., proteins, RNA or DNA which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant polypeptide which is encoded by a nucleic acid incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g, as a cDNA or a genomic or cDNA fragment producedby polymerase chain reaction (PCR) or restriction enzyme digestion)independent of other sequences. It also includes a recombinantpolypeptide which is part of a hybrid polypeptide comprising additionalamino acids. Isolated polypeptides are exemplified by the isolatedkringle, the isolated growth factor domain, and the isolated ATF, whichare described herein.

As used herein, the term “isolated nucleic acid” refers to a nucleicacid segment or fragment which has been separated from sequences whichflank it in a naturally occurring state, e.g., a DNA fragment which hasbeen removed from the sequences which are normally adjacent to thefragment, e.g., the sequences adjacent to the fragment in a genome inwhich it naturally occurs. The term also applies to nucleic acids whichhave been substantially purified from other components which naturallyaccompany the nucleic acid, e.g., RNA or DNA or proteins, whichnaturally accompany it in the cell. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g, asa cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

As used herein, the term “modulating the contractility” or “to modulatethe contractility” of a cell or tissue in the context of the inventivecomposition means to enhance or disinhibit, or to inhibit thecontractility of a cell or tissue, such as, for example, a mammaliansmooth muscle cell or tissue, relative to the contractility of anotherwise identical cell or tissue which is not provided the compositionof the invention or subjected to the method of the invention. Thecontraction process of the cell or tissue in which contractility is tobe evaluated can be either mediated or not mediated by an inducingcompound. When present, the inducing compound is any compound capable ofmediating the contraction of a cell, and includes, by way of example andnot by limitation, neurotransmitter compounds such as phenylepherine(PE), epinepherine, endothelin and acetylcholine (AC).

As used herein, the term “contractility” in the context of a muscle cellor tissue means any one or more of the propensity of the muscle cell ortissue to contract, the force with which the muscle cell or tissuecontracts, the stroke volume of the tissue upon contraction, and thevelocity of muscle tissue shortening (i.e. the extent of shortening of amuscle tissue per unit of time).

As used herein, “to enhance or disinhibit the contractility” of a musclecell or tissue means to increase the contractility of the muscle cell ortissue. By way of example and not by limitation, increasing thecontractility of the muscle cell or tissue can encompass any one or moreof increasing the force of contraction of the muscle cell or tissue,increasing the stroke volume of the muscle tissue during contraction,increasing the propensity of the muscle cell or tissue to contract byincreasing the magnitude or frequency of any signal transduction eventor process associated with promoting contraction (e.g. calcium release),and decreasing the amount of an inducing compound required to mediatecontraction (e.g. decreasing the EC₅₀ of an inducing compound).

As used herein, “to inhibit the contractility” of a muscle cell ortissue means to decrease the contractility of the muscle cell or tissue.By way of example and not by limitation, decreasing the contractility ofthe muscle cell or tissue can encompass any one or more of decreasingthe force of contraction of the muscle cell or tissue, decreasing thestroke volume of the muscle tissue during contraction, decreasing thepropensity of the muscle cell or tissue to contract by decreasing orinhibiting the magnitude or the frequency of any signal transductionevent or process associated with promoting contraction (e.g. calciumrelease), and increasing the amount of an inducing compound required tomediate contraction (e.g. increasing the EC₅₀ of an inducing compound).

As used herein, the term “angiogenic activity” of a cell or tissue inthe context of the methods and compositions of the invention means oneor more of the proliferation and migration of a vascular smooth musclecell involved in the vascularization of a tissue, and the proliferationof an endothelial cell involved in the vascularization of a tissue.

As used herein, the term “modulating angiogenic activity” or “tomodulate the angiogenic activity” or “to modulate angiogenesis” of acell or tissue in the context of the methods and compositions of theinvention means to either promote or to inhibit the angiogenic activityof a vascular smooth muscle cell or tissue or a vascular endothelialcell or tissue. To promote angiogenic activity means to increase orenhance the proliferation or migration of a vascular smooth muscle cellor tissue, or to increase or enhance the proliferation of an endothelialcell involved in the vascularization of a tissue. By way of example andnot by limitation, a compound which inhibits the proliferation of anendothelial cell involved in vascularization of a tissue or whichinhibits the proliferation or migration of a vascular smooth muscle cell(e.g. a compound with an angiostatic activity) modulates the angiogenicactivity of a muscle or endothelial cell or tissue by inhibiting theangiogenic activity. Also by way of example and not by limitation, acompound which promotes the proliferation of an endothelial cellinvolved in vascularization of a tissue or which promotes theproliferation or migration of a vascular smooth muscle cell (e.g. acompound with angiogenic activity) modulates the angiogenic activity ofa muscle or endothelial cell or tissue by promoting or enhancing theangiogenic activity.

As used herein, the term “abnormal angiogenic activity” in the contextof the invention means an unusually high or an unusually low degree ofangiogenic activity in a vascular smooth muscle or vascular endothelialcell or tissue of a mammal relative to an otherwise identical normalvascular smooth muscle or vascular endothelial cell or tissue of amammal, or a degree of angiogenic activity in a vascular smooth muscleor vascular endothelial cell or tissue of a mammal which is deleteriousor disadvantageous to the mammal.

As used herein, the phrase “treating abnormal angiogenic activity” or“treating a disease or condition having abnormal angiogenic activity asa symptom thereof” means to increase or decrease, as appropriate, theangiogenic activity of a vascular smooth muscle cell or tissue or avascular endothelial cell or tissue having an abnormally low orabnormally high angiogenic activity, or having a degree of angiogenicactivity which is deleterious or disadvantageous to the mammal. Thephrase also means to alleviate a symptom of the abnormal angiogenicactivity, or to reduce the severity of a pathological physiologicalconsequence of the abnormal angiogenic activity in the mammal.

By way of example and not by limitation, where the disease or conditionis tumor angiogenesis in a mammal, treating abnormal angiogenic activityin the mammal means to inhibit the endothelial cell proliferation whichis required to develop the tumor neovessels which are to supply thegrowing tumor, lest it outgrow its blood supply. By inhibitingendothelial cell proliferation in this context, the tumor is deprived ofthe vascularization necessary for continued growth, thereby treating thedisease or condition of tumor angiogenesis.

Also, by way of example, and not by limitation, where the disease orcondition is an ocular disorder such as a diabetic or sickle cell orother form of ischemic retinopathy, wherein poorly constructed vascularvessels are induced to grow, but are weak and rupture, leading tobleeding and impaired vision, treating abnormal angiogenic activity inthe mammal means to promote the endothelial cell proliferation which isrequired to develop vascular vessels of proper strength to preventrupture, thereby treating the disease or condition of ischemicretinopathy.

By way of a further example, and not by limitation, where the disease orcondition is any one or more of post-coronary angioplasty, carotidendarterectomy, post-cardiac transplant, and atherosclerosis, whereinthe proliferation of vascular smooth muscle cells in the media andmigration of these cells through the internal elastic lamina into thevascular intima and subsequent proliferation of these cells withconcomitant generation of atherogenic lipids occurs, treating abnormalangiogenic activity in the mammal means to inhibit the vascular smoothmuscle cell proliferation and migration associated with this phenomenon,thereby treating the disease or condition.

As used herein, the term “abnormal muscle cell or tissue contractility,”means a significantly elevated or significantly diminished contractilityof a muscle cell or tissue relative to an otherwise identical normalmuscle cell or tissue. The degree of elevated or diminishedcontractility which is to be considered significant will vary with thespecific type of muscle cell or tissue tested, as well as the specifictype of function evaluated for the muscle cell. The ordinarily skilledartisan will be aware of such variations and the degree of elevated ordiminished contractility to be considered significant. The term alsoincludes spastic contraction of a muscle tissue which is eitherdisadvantageous or deleterious to the mammal or which causes discomfort.The spastic contraction can be either naturally occurring or resultingfrom a surgical procedure.

As used herein, the phrase “treating a disease or condition in a mammalwhich is characterized by abnormal muscle cell or tissue contractility”means to increase or decrease, as appropriate, the contractility of amuscle cell or tissue having an abnormally low or abnormally highcontractility. The phrase also means to reduce the frequency with whichthe abnormal muscle cell or tissue contractility is experienced by apatient having such a disease or condition, to alleviate a symptom ofthe abnormal muscle cell or tissue contractility, or to reduce thefrequency or severity of a pathological physiological consequence of theabnormal muscle cell or tissue contractility in the mammal.

As used herein, the term “recombinant polynucleotide” refers to apolynucleotide having sequences that are not naturally joined together.An amplified or assembled recombinant polynucleotide may be included ina suitable vector, and the vector can be used to transform a suitablehost cell. A recombinant polynucleotide may serve a non-coding function(e.g., promoter, origin of replication, ribosome-binding site, etc.) aswell. A host cell that comprises a recombinant polynucleotide isreferred to as a “recombinant host cell.” A gene which is expressed in arecombinant host cell wherein the gene comprises a recombinantpolynucleotide, expresses a “recombinant polypeptide.”

As used herein, the term “recombinant polypeptide” means a polypeptidewhich is produced upon expression of a recombinant polynucleotide.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

It will be appreciated, of course, that the peptides or polypeptides ofthe invention may incorporate amino acid residues which are modifiedwithout affecting activity. For example, the termini may be derivatizedto include blocking groups, i.e. chemical substituents suitable toprotect and/or stabilize the N- and C-termini from “undesirabledegradation”, a term meant to encompass any type of enzymatic, chemicalor biochemical breakdown of the compound at its termini which is likelyto affect the function of the compound as a modulator of one or more ofcontractility and angiogenic activity of a muscle cell or tissue, i.e.sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Other modifications can also be incorporated without adversely affectingthe biological activity of the peptide for modulating one or more of thecontractility and angiogenic activity of a muscle or endothelial cell ortissue. Such modifications include, but are not limited to, substitutionof one or more of the amino acids in the natural L-isomeric form withamino acids in the D-isomeric form. Thus, the peptide may include one ormore D-amino acid residues, or may comprise amino acids which are all inthe D-form. Retro-inverso forms of peptides in accordance with thepresent invention are also contemplated, for example, inverted peptidesin which all amino acids are substituted with D-amino acid forms.

Acid addition salts of the peptides are also contemplated as functionalequivalents. Thus, a peptide in accordance with the present inventiontreated with an inorganic acid such as hydrochloric, hydrobromic,sulfuric, nitric, phosphoric, and the like, or an organic acid such asan acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic,succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclicand the like, to provide a water soluble salt of the peptide is suitablefor use as a modulator of one or more of the contractility andangiogenic activity of a muscle or endothelial cell or tissue.

The present invention also includes analogs of polypeptides or peptidesof the invention. Analogs can differ from naturally occurring proteinsor peptides by conservative amino acid sequence differences or by any ofthe modifications described above or known in the art which do notaffect sequence, or by both. Modifications can be made as describedabove or by using any technique known to the skilled artisan.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the protein or peptide, do notnormally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a modulator of one or more of thecontractility and angiogenic activity of a muscle or endothelial cell ortissue. Analogs of such polypeptides include those containing residuesother than naturally occurring L-amino acids, e.g. D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

As used herein, an “analog” of a peptide or a polypeptide of theinvention means a peptide or polypeptide which has been modified fromthe naturally occurring peptide or polypeptide by any of themodifications described herein or known to the skilled artisan, butwhich still exhibits activity similar to the naturally occurring peptideor polypeptide as a modulator of one or more of the contractility andangiogenic activity of a mammalian muscle or endothelial cell or tissue.

As used herein, a “chimeric peptide” or “chimeric polypeptide” means apeptide or polypeptide which comprises at least a portion of a naturallyoccurring peptide or polypeptide of the invention and at least a portionof a peptide or polypeptide with which it is normally not found togetherwith in nature, but which still exhibits activity similar to thenaturally occurring peptide or polypeptide of the invention as amodulator of one or more of the contractility and angiogenic activity ofa mammalian muscle or endothelial cell or tissue.

As used herein, a “functional element” of a peptide or a polypeptide ofthe invention means a portion of a peptide or polypeptide of theinvention which participates in the modulation of one or more of thecontractility and angiogenic activity of a mammalian muscle orendothelial cell or tissue. A method of identifying a functional elementof a peptide or polypeptide of the invention is described herein.

As used herein, an “epitope” of a peptide or a polypeptide of theinvention means a portion of a peptide or polypeptide of the inventionwhich is exposed at the surface of the peptide or polypeptide of theinvention or which is accessible to a binding protein of a peptide orpolypeptide of the invention. A method of identifying a binding proteinof a peptide or polypeptide of the invention is described herein.

As used herein, a “binding protein” of a peptide or polypeptide of theinvention means a protein or polypeptide which substantially binds tothe peptide or polypeptide of the invention, but which does notsubstantially bind to other peptides or proteins which are associatedwith the peptide or polypeptide of the invention, wherein the “bindingprotein” also transmits a biochemical signal or participates in a signaltransduction event which mediates the contraction of a muscle cell orwhich mediates the angiogenic activity of a muscle or endothelial cell.The binding protein can also participate in signal transductionindirectly, by facilitating the interaction of the peptide orpolypeptide of the invention with, or by acting in concert with anothermolecule which provides the proximate signal which mediates muscle cellcontraction or muscle or endothelial cell angiogenic activity. By way ofexample and not by limitation, the binding protein can be a receptor ora transport protein of the peptide or polypeptide of the invention. Thebinding protein can be any binding protein known or yet to be known, andincludes, by way of example and not by limitation, certainbeta-integrins as binding proteins which participate in signaltransduction directly, and the low-density lipoprotein related proteinor the alpha 2 macroglobulin receptor (LRP/α2 macroglobulin receptor) asa binding protein which participates in signal transduction indirectlyby down-regulating the signal transduction process.

As used herein, the term “vector” means a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into cells, such as, for example, polylysine compounds,liposomes, and the like. Examples of viral vectors include, but are notlimited to, adenoviral vectors, adeno-associated viral vectors,retroviral vectors, and the like.

As used herein, the term “expression vector” refers to a vectorcomprising a recombinant polynucleotide comprising expression controlsequences operatively linked to a nucleotide sequence to be expressed.An expression vector comprises sufficient cis-acting elements forexpression; other elements for expression can be supplied by the hostcell or in an in vitro expression system. Expression vectors include allthose known in the art, such as cosmids, plasmids (e.g., naked orcontained in liposomes) and viruses that incorporate the recombinantpolynucleotide.

Description

The invention includes compositions comprising the uPA kringle or thegrowth factor domain alone, or in combination with one or more otherdomains of the uPA molecule, in an amount effective to modulate one ormore of the contractility of a mammalian muscle cell or tissue and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.The compositions of the invention are useful for the treatment of adisease or condition in a mammal having as a symptom thereof one or moreof abnormal muscle cell or tissue contractility and abnormal muscle orendothelial cell or tissue angiogenic activity. The domain or domains ofuPA can be present in the inventive compositions and methods either asan isolated polypeptide, as part of the whole uPA molecule in eithersingle chain or two chain form (scuPA or tcuPA, respectively), or aspart of a deletion mutant thereof, which lacks either the uPA kringle orthe connecting peptide.

The inventive method comprises administering to a mammal afflicted withsuch a disease or condition an effective amount of an inventivecomposition, and modulating the contractility and/or the angiogenicactivity of the muscle or endothelial cell or tissue, thereby treatingthe disease or condition. The invention also includes methods foridentifying and using binding proteins, agonists and antagonists for oneor more domains of uPA to modulate muscle cell or tissue contractilityand/or angiogenic activity, as well as using agonists and antagonists tothe binding proteins themselves to modulate muscle or endothelial cellor tissue contractility and/or angiogenic activity, thereby treating thedisease or condition.

In the methods and compositions of the invention, the disease orcondition can be any disease or condition in a mammal in which one ormore of abnormal muscle cell or tissue contractility and abnormalangiogenic activity is a symptom. Such diseases and conditions include,by way of example and not by limitation, the following: cardiovasculardiseases and conditions such as hypotension, hypertension andatherosclerosis; conditions which promote thrombotic disorders such asstroke, heart attack, microvascular occlusions, thromboticmicroangiopathies, and surgically induced thrombotic disorders (i.e.post angioplasty stenting); angiogenic disorders; respiratory diseasesand conditions such as pulmonary fibrosis and asthma; invasion,angiogenesis and metastasis of cancer or tumor cells; ocular disorderssuch as glaucoma and diabetic retinopathy; wound healing disorders,disorders of fibrinolysis and clotting; and reproductive disorders suchas uterine contraction disorders and male impotence.

In the methods and compositions of the invention, the cell can be anytype of mammalian muscle cell or vascular endothelial cell. Types ofmuscle cells include a smooth muscle cell, a striated muscle cell and acardiac muscle cell. Preferably, the cell is a smooth muscle cell.Examples of preferred smooth muscle cells include vascular and bronchialsmooth muscle cells.

In preferred embodiments of the invention, the mammalian muscle orendothelial cell is a part of a mammalian muscle tissue, and is in amammal. Preferably, the inventive composition comprises the one or moredomains of uPA in an amount effective to modulate the contractilityand/or angiogenic activity of a mammalian muscle or endothelial tissue.

The invention includes a composition comprising the uPA kringle in anamount effective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.The uPA kringle is a polypeptide portion of the urokinase-typeplasminogen activator protein which shares at least about 75% homologywith the polypeptide corresponding to SEQ ID NO:1 (FIG. 1A). Preferably,the uPA kringle is about 80% homologous, more preferably about 85%homologous, even more preferably 90% homologous, yet more preferably 95%homologous, and most preferably about 99% homologous to the polypeptidecorresponding to SEQ ID NO:1. Even more preferably, the uPA kringle isthe polypeptide corresponding to SEQ ID NO:1.

The determination of percent homology (i.e. percent identity) describedherein between two amino acid or nucleotide sequences can beaccomplished using a mathematical algorithm. For example, a mathematicalalgorithm useful for comparing two sequences is the algorithm of Karlinand Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modifiedas in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA90:5873-5877). This algorithm is incorporated into the NBLAST and XBLASTprograms of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and canbe accessed, for example, at the National Center for BiotechnologyInformation (NCBI) world wide web site having the universal resourcelocator “http://www.ncbi.nlm.nih.gov/BLAST/”. BLAST nucleotide searchescan be performed with the NBLAST program (designated “blastn” at theNCBI web site), using the following parameters: gap penalty=5; gapextension penalty=2; mismatch penalty=3; match reward=1; expectationvalue 10.0; and word size=11 to obtain nucleotide sequences homologousto a nucleic acid described herein. BLAST protein searches can beperformed with the XBLAST program (designated “blastn” at the NCBI website) or the NCBI “blastp” program, using the following parameters:expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acidsequences homologous to a protein molecule described herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The percent identity between two amino acid or nucleotide sequences canbe determined using techniques similar to those described above, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

In a preferred embodiment, the uPA kringle is present in an amounteffective to enhance or disinhibit the contractility of a mammaliansmooth muscle cell or tissue. In another preferred embodiment, the uPAkringle is present in an amount effective to inhibit the angiogenicactivity of a vascular smooth muscle cell or tissue or a vascularendothelial cell or tissue.

Preferably, the uPA kringle is present in the inventive composition at aconcentration ranging from about ten picomolar to about one hundredmicromolar.

In one embodiment, the uPA kringle is present in the inventivecomposition as an isolated kringle. An isolated kringle is an isolatedpolypeptide which comprises the uPA kringle. The isolated kringle can beprepared by any method known to the skilled artisan for preparing anisolated polypeptide.

For example, the isolated kringle can be obtained by preparing andpurifying a recombinant version of any of the polypeptides describedherein which comprise the uPA kringle. Molecular biology techniques forthe preparation of recombinant polypeptides are well known in the art,and are described for example in Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York;Ausubel et al., 1994, Current Protocols in Molecular Biology, John Wiley& Sons, New York), and Gerhardt et al., eds., 1994, Methods for Generaland Molecular Bacteriology, American Society for Microbiology,Washington, D.C. Protein purification methods are also well known in theart, and are described, for example in Deutscher et al. (ed., 1990,Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

In a preferred aspect, the isolated kringle is prepared by performing alimited proteolysis of scuPA, then purifying the resulting polypeptideusing reverse-phase HPLC, as described herein in the Examples.

The invention also includes a composition comprising the uPA growthfactor domain in an amount effective to modulate the contractilityand/or the angiogenic activity of a mammalian muscle or endothelialcell. The growth factor domain is a polypeptide portion of theurokinase-type plasminogen activator protein which shares at least about75% homology with the polypeptide corresponding to SEQ ID NO:2 (FIG.1B). Preferably, the growth factor domain is about 80% homologous, morepreferably about 85% homologous, even more preferably 90% homologous,yet more preferably 95% homologous, and most preferably about 99%homologous to the polypeptide corresponding to SEQ ID NO:2. Even morepreferably, the growth factor domain is the polypeptide corresponding toSEQ ID NO:2.

In a preferred embodiment, the uPA growth factor domain is present in anamount effective to inhibit the contractility of a mammalian smoothmuscle cell or tissue. Preferably, the uPA growth factor domain ispresent in the inventive composition at a concentration ranging fromabout 10 picomolar to about 100 micromolar.

In one embodiment, the growth factor domain is present in the inventivecomposition as an isolated growth factor domain. An isolated growthfactor domain is an isolated polypeptide which comprises the uPA growthfactor domain. The isolated growth factor domain can be prepared by anymethod known to the skilled artisan for preparing an isolatedpolypeptide. For example, the isolated growth factor domain can beobtained by preparing and purifying a recombinant version of any of thepolypeptides described herein which comprise the growth factor domain.Molecular biology techniques for the preparation of recombinantpolypeptides are well known in the art, and are discussed above.

In other embodiments, the inventive composition comprises, in additionto the uPA kringle or the growth factor domain, one or more additionaldomains of the uPA protein selected from the group consisting of theconnecting peptide, the protease domain, the uPA kringle and the growthfactor domain.

In one aspect, the composition of the invention comprises the full uPAprotein. The full uPA protein comprises, from the N-terminal end to theC-terminal end, respectively, the growth factor domain, the kringle, theconnecting peptide, and the protease domain. The full uPA protein is apolypeptide which shares at least about 75% homology with thepolypeptide corresponding to SEQ ID NO:3 (FIG. 1C). Preferably, the fulluPA protein is about 80% homologous, more preferably about 85%homologous, even more preferably 90% homologous, yet more preferably 95%homologous and most preferably about 99% homologous to the polypeptidecorresponding to SEQ ID NO:3. Even more preferably, the full uPA proteinis the polypeptide corresponding to SEQ ID NO:3.

The inventive composition can comprise the full uPA protein in nascentform, which is termed single chain urokinase (scuPA). Single chainurokinase (scuPA) is the form of the urokinase-type plasminogenactivator protein which is synthesized by mammalian cells as the parentmolecule. In this embodiment, the growth factor domain of scuPA ispresent in an amount effective to inhibit the contractility of amammalian muscle cell. Preferably, the growth factor domain is presentin an amount effective to cause a dose-dependent inhibition ofacetylcholine induced contraction of a bronchial smooth muscle cell.

The molecule scuPA is capable of being converted by either anautoactivation or by a protease to a form termed two chain urokinase(tcuPA). Proteases capable of converting scuPA to tcuPA include plasminand kallikrein. The two chain form of urokinase is produced byproteolytic cleavage of scuPA between amino acid residues number 158 and159 of the full uPA protein, and the resulting tcuPA molecule is heldtogether in two chain form by disulfide bonds.

In another aspect, the inventive composition comprises tcuPA, apolypeptide which shares at least about 75% homology with thepolypeptide corresponding to SEQ ID NO:3 (FIG. 1C). Preferably, tcuPA isabout 80% homologous, more preferably about 85% homologous, even morepreferably 90% homologous, yet more preferably 95% homologous, and mostpreferably about 99% homologous to the polypeptide corresponding to SEQID NO:3. Even more preferably, tcuPA is the polypeptide corresponding toSEQ ID NO:3. In this embodiment, tcuPA is present in an amount effectiveto modulate the contractility of a mammalian muscle cell.

In one preferred aspect, the uPA kringle is present in the inventivecomposition in an amount effective to enhance or disinhibit PE-inducedcontraction of a mammalian vascular smooth muscle cell or tissue.

In another preferred aspect, the uPA kringle is present in the inventivecomposition in an amount effective to inhibit AC-induced contraction ofa mammalian bronchial smooth muscle cell or tissue.

In another embodiment, the inventive composition comprises the aminoterminal fragment of uPA (ATF). The ATF is a polypeptide portion of thefull uPA protein which comprises the growth factor domain and the uPAkringle. The ATF shares at least about 75% homology with the polypeptidecorresponding to SEQ ID NO:4 (FIG. 1D). Preferably, the ATF is about 80%homologous, more preferably about 85% homologous, even more preferably90% homologous, yet more preferably 95% homologous, and most preferablyabout 99% homologous to the polypeptide corresponding to SEQ ID NO:4.Even more preferably, the ATF is the polypeptide corresponding to SEQ IDNO:4.

In embodiments of the inventive composition comprising the ATF, the ATFis present in an amount effective to modulate the contractility orangiogenic activity of a mammalian muscle or endothelial cell or tissue.Preferably, the uPA kringle of ATF is present in the inventivecomposition in an amount effective to enhance or disinhibit PE-inducedcontraction of a mammalian vascular smooth muscle cell or tissue or inan amount effective to inhibit the angiogenic activity of a vascularsmooth muscle cell or tissue or a vascular endothelial cell or tissue.

In one embodiment, the ATF is present in the inventive composition as anisolated ATF. An isolated ATF is an isolated polypeptide which comprisesthe uPA kringle and the uPA growth factor domain. The isolated ATF canbe prepared by any method known to the skilled artisan for preparing anisolated polypeptide. For example, the isolated ATF can be obtained bypreparing and purifying a recombinant version of any of the polypeptidesdescribed herein which comprise the ATF. Molecular biology techniquesfor the preparation of recombinant polypeptides are well known in theart, and are discussed above.

Preferably, the isolated ATF is prepared by autodigestion of tcuPA. Inanother preferred aspect, where the polypeptide of the inventioncomprises both the ATF and the connecting peptide, the polypeptide isprepared by matrilysin (MMP-7) or stromelysin (MMP-3) digestion of uPA.

In any of the compositions and methods of the invention describedherein, a functional element, an analog, an epitope, or a chimericpeptide can be used in place of one or more of the isolated uPA kringle,the isolated growth factor domain and the isolated ATF. This enables theuse of a smaller polypeptide in the inventive compositions havingsimilar or greater activity than the corresponding larger isolatedpolypeptide for modulating muscle cell contractility or angiogenicactivity. Methods for identifying a functional element, an epitope, ananalog or a chimeric polypeptide for any of these isolated polypeptidesare described herein and known in the art. By way of example and not bylimitation, such functional elements, analogs, epitopes and chimericpeptides can be prepared using recombinant technology or isolated fromnatural sources. Alternatively, they can be prepared synthetically byusing any polypeptide synthesis method known in the art, such as asolid-phase polypeptide synthesis method.

The invention also includes a composition comprising a polypeptide whichcomprises the connecting peptide and the protease domains of uPA, in anamount effective to modulate the contractility of a mammalian musclecell or tissue. This polypeptide is termed LMW-uPA, and shares at leastabout 75% homology with the polypeptide corresponding to SEQ ID NO:5(FIG. 1E). Preferably, the LMW-uPA is about 80% homologous, morepreferably about 85% homologous, even more preferably 90% homologous,yet more preferably 95% homologous, and most preferably about 99%homologous to the polypeptide corresponding to SEQ ID NO:5. Even morepreferably, the LMW-uPA is the polypeptide corresponding to SEQ ID NO:5.

In a preferred embodiment, the LMW-uPA is present in the inventivecomposition in an amount effective to inhibit the contractility of amammalian smooth muscle cell or tissue.

The invention also includes a composition comprising a polypeptidedeletion mutant of either scuPA or tcuPA in which the connecting peptidehas been deleted. Methods for preparing deletion mutants of wild typepolypeptides are known in the art, and a preferred method is describedherein in the Examples.

In one embodiment, a uPA deletion mutant lacking the connecting peptide(scuPA^(Δ136-143)) is present in the inventive composition in an amounteffective to modulate the contractility and/or the angiogenic activityof a mammalian muscle or endothelial cell. The scUPA^(Δ136-143) sharesat least about 75% homology with the polypeptide corresponding to SEQ IDNO:6 (FIG. 1F). Preferably, the uPA deletion mutant is about 80%homologous, more preferably about 85% homologous, even more preferably90% homologous, yet more preferably 95% homologous and most preferablyabout 99% homologous to the polypeptide corresponding to SEQ ID NO:6.Even more preferably, the uPA deletion mutant is the polypeptidecorresponding to SEQ ID NO:6.

In a preferred embodiment, scuPA^(Δ136-143) is present in the inventivecomposition in an amount effective to enhance or disinhibit thecontractility of a mammalian smooth muscle cell.

The invention also includes a composition comprising a polypeptidedeletion mutant of either scuPA or tcuPA in which the uPA kringle hasbeen deleted. Methods for preparing deletion mutants of wild typepolypeptides are known in the art, and a preferred method is describedherein in the Examples.

In one embodiment, a uPA deletion mutant of scuPA or tcuPA lacking theuPA kringle (Δkringle-scuPA or Δkringle-tcuPA) is present in theinventive composition in an amount effective to modulate thecontractility and/or the angiogenic activity of a mammalian muscle orendothelial cell. The Δkringle-scuPA shares at least about 75% homologywith-the polypeptide corresponding to SEQ ID NO:7 (FIG. 1G). Preferably,the uPA deletion mutant is about 80% homologous, more preferably about85% homologous, even more preferably 90% homologous, yet more preferably95% homologous and most preferably about 99% homologous to thepolypeptide corresponding to SEQ ID NO:7. Even more preferably, the uPAdeletion mutant is the polypeptide corresponding to SEQ ID NO:7.

In a preferred embodiment, one or more of Δkringle-scuPA andΔkringle-tcuPA is present in the inventive composition in an amounteffective to inhibit the contractility of a mammalian smooth muscle celland to proteolytically activate plasminogen.

In another embodiment, the inventive composition comprises a polypeptidecomprising the ATF and the connecting peptide in an amount effective tomodulate one or more of the contractility and angiogenic activity of amammalian muscle or endothelial cell or tissue. In this aspect, thepolypeptide comprising the ATF and the connecting peptide shares atleast about 75% homology with the polypeptide corresponding to SEQ IDNO:8 (FIG. 1H). Preferably, the polypeptide is about 80% homologous,more preferably about 85% homologous, even more preferably 90%homologous, yet more preferably 95% homologous, and most preferablyabout 99% homologous to the polypeptide corresponding to SEQ ID NO:8.Even more preferably, the polypeptide is the polypeptide correspondingto SEQ ID NO:8. Without wishing to be bound by any one theory, it issuspected that the connecting peptide domain, when present in the samepolypeptide with the ATF, results in a polypeptide which is moreeffective at modulating angiogenic activity than the ATF alone, possiblyby affecting cell adhesion.

The invention also includes a composition comprising a polypeptide whichcomprises the uPA kringle and the connecting peptide, in an amounteffective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.This polypeptide shares at least about 75% homology with the polypeptidecorresponding to SEQ ID NO:9 (FIG. 1I). Preferably, the polypeptide isabout 80% homologous, more preferably about 85% homologous, even morepreferably 90% homologous, yet more preferably 95% homologous, and mostpreferably about 99% homologous to the polypeptide corresponding to SEQID NO:9. Even more preferably, the polypeptide is the polypeptidecorresponding to SEQ ID NO:9.

In another embodiment, any of the compositions of the inventiondescribed above can further comprise an inducing compound present in anamount effective to mediate contraction of a mammalian muscle cell. Theinducing compound can be any compound capable of mediating thecontraction of a mammalian muscle cell, and includes, by way of exampleand not by limitation, neurotransmitter compounds such as phenylepherine(PE), acetylcholine (AC), epinepherine and endothelin. The amount ofsuch compounds to be effective for mediating contraction of a mammalianmuscle cell will be apparent to the skilled artisan.

In another embodiment, the inventive composition comprises any of thecompositions of the invention described above, and further comprises oneor more compounds suitable for one or more of promoting, enhancing,prolonging and amplifying the effectiveness of the composition of theinvention for modulating the contractility and/or the angiogenicactivity of a mammalian muscle or endothelial cell or tissue. In oneaspect, the one or more compounds are selected from the group consistingof agonists, antagonists and binding proteins of any one or more domainsof uPA. Methods of identifying agonists, antagonists and bindingproteins to one or more domains of uPA are described herein. Also, theone or more compounds can be agonists or antagonists to the bindingproteins themselves.

The invention also includes an isolated nucleic acid encoding apolypeptide comprising one or more of the domains of uPA describedherein. The isolated nucleic acid shares at least about 75% homology,preferably about 80% homology, more preferably about 85% homology, evenmore preferably about 90% homology, yet more preferably 95% homology,and most preferably about 99% homology with a nucleic acid selected fromthe group consisting of SEQ ID NO:10 (FIG. 1J), SEQ ID NO:1 (FIG. 1K),SEQ ID NO:12 (FIG. 1L), SEQ ID NO:13 (FIG. 1M), SEQ ID NO:14 (FIG. 1N),SEQ ID NO:15 (FIG. 1O), SEQ ID NO:16 (FIG. 1P), SEQ ID NO:17 (FIG. 1Q)and SEQ ID NO:18 (FIG. 1R). Even more preferably, the nucleic acid isselected from the group consisting of SEQ ID NO:10-SEQ ID NO:18. In thisembodiment of the invention, the isolated nucleic acid is present in anamount effective to transform a mammalian muscle or endothelial cell toprovide transgene expression of the one or more polypeptide domains ofuPA at a level of expression effective to modulate the contractilityand/or angiogenic activity of the mammalian muscle or endothelial cellso transfected.

The isolated nucleic acid can be either alone as a “naked” nucleic acid,such as a linearized nucleic acid, or as a component of any type ofvector suitable for transfecting a mammalian muscle or endothelial celldescribed herein or known in the art. Preferably, the isolated nucleicacid is a recombinant polynucleotide component of a viral or plasmidexpression vector suitable for transfecting a mammalian muscle orendothelial cell, and is operably linked to the appropriate regulatoryelements to provide a high level of expression of the transgene once atargeted mammalian muscle or endothelial cell is transformed with theisolated nucleic acid. Examples of preferred vectors include adenovirus,retrovirus, lentivirus and adeno-associated virus vectors. Techniquesfor using such vectors to transfect a mammalian muscle or endothelialcell are known in the art.

When any one of the polypeptides described herein comprising one or moredomains of uPA are to be administered to a mammal or to a tissue of amammal for the purpose of exerting a beneficial effect in the mammal,the invention should be construed to include delivery of the polypeptidevia delivery of an isolated nucleotide sequence encoding the peptide.Expression of the peptide from the nucleotide sequence so delivered tothe desired tissue is effective administration of the peptide to thetissue.

In another embodiment, the inventive composition is in the form of apharmaceutical composition comprising a pharmaceutically acceptablecarrier. Such a pharmaceutical composition may consist of the inventivecomposition alone as the active ingredient, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the inventive composition as the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of apharmaceutically acceptable salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “pharmaceutically acceptable salt” means a saltform of the active ingredient which is compatible with any otheringredients of the pharmaceutical composition, which is not deleteriousto the subject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to any mammal. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various mammals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor any route of administration known to the skilled artisan, including,by way of example and not by limitation, oral, parenteral, topical,ocular, inhalation, intrauterine, intravesicular, intraurethral andbuccal routes of administration. The pharmaceutical composition can beadministered to a mammal by any route of administration known to theskilled artisan, such as those described above, and by any method ofadministering a pharmaceutical composition to a mammal known in the art.A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may farther comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the composition of the invention which may beadministered to an animal, preferably a human, range in amount from 1microgram to about 100 grams per kilogram of body weight of the animal.While the precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal and the routeof administration. Preferably, the dosage of the composition will varyfrom about 1 mg to about 10 g per kilogram of body weight of the animal.More preferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The composition may be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenless frequently, such as once every several months or even once a yearor less. The frequency of the dose will be readily apparent to theskilled artisan and will depend upon any number of factors, such as, butnot limited to, the type and severity of the disease being treated, thetype and age of the animal, etc.

The invention also includes a method of treating a disease or conditionin a mammal which has as a symptom thereof one or more of abnormalmuscle cell or tissue contractility and abnormal muscle or endothelialcell or tissue angiogenic activity. The components used in the method ofthe invention are the same as those described above in the inventivecompositions. The mammal can be any mammal described in the inventivecompositions, but is preferably a human. The muscle or endothelial cellor tissue can be any of the mammalian cell or tissue types described inthe inventive compositions. A preferred muscle cell is a smooth musclecell.

The diseases or conditions in a mammal which can be treated by themethod of the invention are the same diseases and conditions describedabove in the inventive compositions. By way of example and not bylimitation, the method of the invention can be used to treatcardiovascular diseases and conditions such as hypotension, hypertensionand atherosclerosis; conditions which promote thrombotic disorders suchas stroke, heart attack, microvascular occlusions, thromboticmicroangiopathies, and surgically induced thrombotic disorders (i.e.post angioplasty stenting); angiogenic disorders; respiratory diseasesand conditions such as pulmonary fibrosis and asthma; invasion,angiogenesis and metastasis of cancer or tumor cells; ocular disorderssuch as glaucoma and diabetic retinopathy; wound healing disorders,disorders of fibrinolysis and clotting; and reproductive disorders suchas uterine contraction disorders and male impotence.

In the method of the invention, any of the inventive compositions can beadministered to the mammal to be treated by any route of administrationknown in the art or described herein. In one aspect, the inventivecomposition is administered to the mammal in the form of apharmaceutical composition. The pharmaceutical composition can be any ofthe pharmaceutical compositions described herein in the inventivecompositions.

In the method of the invention, any of the compositions of the inventionmay comprise an inducer compound in an amount effective to mediate thecontraction of a mammalian muscle cell. The inducer compound can be anyof those described in the inventive compositions.

Also, in the method of the invention, any of the compositions of theinvention may further comprise one or more compounds known in the art toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, resealed erythrocytes, andimmunologically based systems such as antibody targeting systems mayalso be used to administer the inventive composition according to themethods of the invention.

The method comprises administering to a mammal afflicted with such adisease or condition a composition comprising the uPA kringle in anamount effective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.In one aspect, the uPA kringle shares at least about 75% homology with apolypeptide having the amino acid sequence corresponding to SEQ ID NO:1.The uPA kringle component of the inventive composition can be obtainedfrom any of the natural or recombinant sources described herein in theinventive compositions.

In one embodiment, the composition administered to the mammal comprisesthe uPA kringle as an isolated kringle in an amount effective tomodulate one or more of the contractility and the angiogenic activity ofa mammalian muscle or endothelial cell or tissue. The isolated kringlecan be prepared by any method described herein or known to the skilledartisan.

In another embodiment, the composition administered to the mammalcomprises the uPA kringle as part of a polypeptide which shares at leastabout 75% homology with a polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:3 (tcuPA), SEQ ID NO:4(ATF), SEQ ID NO:6 (scuPA^(Δ136-143)), SEQ ID NO:8 and SEQ ID NO:9. TheuPA kringle is present in the composition in an amount effective tomodulate one or more of the contractility and the angiogenic activity ofa mammalian muscle or endothelial cell or tissue.

In yet another embodiment, the composition comprises, in place of theuPA kringle, one or more of a functional element thereof, an epitopethereof, an analog thereof and a chimeric polypeptide thereof, in anamount effective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell.

In a further embodiment, the composition of the invention administeredto the mammal further comprises, in addition to the uPA kringle (or theone or more functional element thereof, epitope thereof, analog thereofand chimeric polypeptide thereof), one or more of an agonist of the uPAkringle, an agonist of a binding protein of the uPA kringle, anantagonist of the uPA growth factor domain, an antagonist of theconnecting peptide, an antagonist of a binding protein of the uPA growthfactor domain, and an antagonist of a binding protein of the connectingpeptide. These agonists, antagonists and binding proteins are describedherein below.

The inventive method also includes modulating one or more of thecontractility and the angiogenic activity of the muscle or endothelialcell or tissue having one or more of abnormal contractility and abnormalangiogenic activity. The contractility and/or angiogenic activity of themuscle or endothelial cell or tissue can be either enhanced,disinhibited, or inhibited, relative to an otherwise identical muscle orendothelial cell or tissue in the mammal which is not provided theinventive composition. By modulating one or more of the contractilityand the angiogenic activity of the muscle or endothelial cell or tissue,the disease or condition in the mammal is treated.

By way of example and not by limitation, where the disease or conditiontreated is tumor angiogenesis in a mammal, the angiogenic activity inthe mammal is modulated by inhibiting the endothelial cell proliferationwhich is required to develop the tumor neovessels which are to supplythe growing tumor, lest it outgrow its blood supply. By inhibitingendothelial cell proliferation in this context, the tumor is deprived ofthe vascularization necessary for continued growth, thereby treating thedisease or condition of tumor angiogenesis.

Also, by way of example, and not by limitation, where the disease orcondition treated is an ocular disorder such as a diabetic or sicklecell or other form of ischemic retinopathy, wherein poorly constructedvascular vessels are induced to grow, but are weak and rupture, leadingto bleeding and impaired vision, the angiogenic activity in the mammalis modulated by enhancing the endothelial cell proliferation which isrequired to develop vascular vessels of proper strength to preventrupture, thereby treating the disease or condition of ischemicretinopathy.

By way of a further example, and not by limitation, where the disease orcondition treated is any one or more of post-coronary angioplasty,carotid endarterectomy, post-cardiac transplant, and atherosclerosis,wherein the proliferation of vascular smooth muscle cells in the mediaand migration of these cells through the internal elastic lamina intothe vascular intima and subsequent proliferation of these cells withconcomitant generation of atherogenic lipids occurs, the angiogenicactivity in the mammal is modulated by inhibiting the vascular smoothmuscle cell proliferation and migration associated with this phenomenon,thereby treating the disease or condition.

Also by way of example, and not by limitation, where the disease orcondition in the mammal sought to be prevented is congestive heartfailure (CHF), the contractility of cardiac muscle tissue is modulatedby inhibiting tonic contraction of cardiac muscle tissue, therebypreventing the development of CHF. In contrast, where CHF has alreadydeveloped, the CHF is treated by enhancing the contractility of cardiacmuscle tissue in order to increase the stroke volume upon contraction ofthe cardiac muscle tissue.

A further non-limiting example, where the disease or condition treatedin the mammal is either an inherited or an acquired dystrophic conditionof a skeletal muscle tissue, the contractility of the skeletal muscletissue is modulated by enhancing the contractility of the skeletalmuscle tissue in order to increase or preserve muscle power, therebytreating the disease. In contrast, where the disease or conditiontreated involves skeletal muscle spasms which are deleterious,disadvantageous, or create discomfort in a mammal, the contractility ofthe affected skeletal muscle tissue is modulated by inhibiting thecontractility of the skeletal muscle tissue.

In the following additional non-limiting examples, where the disease orcondition treated in the mammal has as a symptom thereof abnormally highcontractility of a smooth muscle cell or tissue, the contractility ismodulated by inhibiting the contractility of the affected smooth musclecell or tissue. By way of example and not by limitation, for abnormaluterine contraction such as premature labor, the contractility ofuterine smooth muscle tissue is inhibited; for male impotence, thecontractility of vascular smooth muscle tissue is inhibited to promotevasodilation; for angina, the contractility of vascular smooth muscletissue is inhibited to promote vasodilation; for any type of inheritedor acquired cause of thrombosis associated with premature fetal demiseor spontaneous abortion, wherein the pathophysiology involvesthrombosis/vascular contraction (including eclampsia), the contractilityof vascular smooth muscle tissue is inhibited to promote vasodilationand to increase blood flow to the fetus; for asthma or for primary orsecondary pulmonary hypertension, the contractility of vascular smoothmuscle tissue is inhibited to promote vasodilation; for enhancingdelivery of a thrombolytic agent to any part of the cardiovascularsystem of a mammal the contractility of vascular smooth muscle tissue isinhibited to promote vasodilation; and for inhibiting the spasticcontraction of vascular smooth muscle tissue after vascular interventionsuch as after angioplasty, endarterectomy, thrombectomy or othervascular interventions, the contractility of vascular smooth muscletissue is inhibited to reduce the frequency and intensity of the spasticcontractions and to promote vasodilation.

In one preferred embodiment, the uPA kringle or functional elementthereof, epitope thereof, analog thereof or chimeric polypeptide thereofis administered to the mammal in an amount effective to enhance ordisinhibit the contractility of a mammalian smooth muscle cell or tissueand/or in an amount effective to inhibit the angiogenic activity of avascular smooth muscle or vascular endothelial cell or tissue.Preferably, the smooth muscle cell or tissue is a vascular smooth musclecell or tissue and the disease or condition treated is one or more ofhypotension and cancer or tumor cell invasion, angiogenesis, growth andmetastasis.

The invention also includes a method for treating a mammal afflictedwith a disease or condition having as a symptom thereof one or more ofabnormal contractility and abnormal angiogenic activity. The methodcomprises administering to the mammal a composition comprising the uPAgrowth factor domain in an amount effective to modulate one or more ofthe contractility and the angiogenic activity of a mammalian muscle orendothelial cell or tissue. In one aspect, the uPA growth factor domainshares at least about 75% homology with a polypeptide having the aminoacid sequence corresponding to SEQ ID NO:2. The uPA growth factor domaincomponent of the inventive composition can be obtained from any of thenatural or recombinant sources described herein in the inventivecompositions.

In one embodiment, the composition administered to the mammal comprisesthe uPA growth factor domain as an isolated growth factor domain in anamount effective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.The isolated growth factor domain can be prepared by any methoddescribed herein or known to the skilled artisan.

In another embodiment, the composition administered to the mammalcomprises the uPA growth factor domain as part of a polypeptide whichshares at least about 75% homology with a polypeptide having the aminoacid sequence selected from the group consisting of SEQ ID NO:3 (scuPA),SEQ ID NO:4 (ATF), SEQ ID NO:6 (scUPA^(Δ136-143)), SEQ ID NO:7(Δkringle-scuPA or Δkringle-tcuPA) and SEQ ID NO:8). The uPA growthfactor domain is present in the polypeptide in an amount effective tomodulate one or more of the contractility and the angiogenic activity ofa mammalian muscle or endothelial cell or tissue.

In yet another embodiment, the composition comprises, in place of theuPA growth factor domain, one or more of a functional element thereof,an epitope thereof, an analog thereof and a chimeric polypeptidethereof, in an amount effective to modulate one or more of thecontractility and the angiogenic activity of a mammalian muscle orendothelial cell or tissue.

In a further embodiment, the composition of the invention administeredto the mammal further comprises, in addition to the uPA growth factordomain (or the one or more functional element thereof, epitope thereof,analog thereof and chimeric polypeptide thereof), one or more of anagonist of the uPA growth factor domain, an agonist of the connectingpeptide, an agonist of a binding protein of the growth factor domain, anagonist of a binding protein of the connecting peptide, an antagonist ofthe uPA kringle, and an antagonist of a binding protein of the uPAkringle. These agonists, antagonists and binding proteins are describedherein below.

The inventive method also includes one or more of modulating thecontractility and modulating the angiogenic activity of the muscle orendothelial cell or tissue having one or more of abnormal contractilityand abnormal angiogenic activity. The contractility of the muscle cellor tissue can be either inhibited, disinhibited or enhanced, relative toan otherwise identical muscle cell or tissue in the mammal which is notprovided the inventive composition. By modulating one or more of thecontractility and the angiogenic activity of the muscle or endothelialcell or tissue, the disease or condition in the mammal is treated.

In one preferred aspect, the uPA growth factor domain or functionalelement thereof, epitope thereof, analog thereof or chimeric polypeptidethereof is administered to the mammal in an amount effective to inhibitthe contractility of a mammalian smooth muscle cell or tissue and/or tomodulate the angiogenic activity of the smooth muscle or endothelialcell. In one preferred aspect, the smooth muscle cell or tissue is avascular smooth muscle cell or tissue and the disease or conditiontreated is hypertension.

Optionally, the inventive method can include providing the inventivecomposition to the interior of a muscle or endothelial cell or tissuehaving abnormal contractility and/or abnormal angiogenic activity. Theinventive composition can be provided to the cell or tissue either alonein “naked” form, for example as an isolated polypeptide, or formulatedin a vehicle suitable for delivery, such as, by way of example and notby limitation, in a complex with a cationic molecule or a liposomeforming lipid, in a vector, or as a component of a pharmaceuticalcomposition. Such vehicles are well known to the skilled artisan.

The inventive composition can be provided to the cell either directly,by contacting the inventive composition with the cell sought to bemodulated, or indirectly, such as through the action of any biologicalprocess. By way of example and not by limitation, the inventivecomposition can be provided to the cell by using a liposome, bycontacting the composition with the cell, by transfecting the cell usinga vector, by injecting the composition into a tissue or fluidsurrounding the cell, by simple diffusion of the composition across thecell membrane, or by any active or passive transport mechanism acrossthe cell membrane. In addition, any of the methods known in the art forproviding targeted or in situ delivery of the inventive composition tothe interior of a desired cell or to a targeted tissue can be used. Byway of example and not by limitation, antibody targeting is one of suchmethods.

In embodiments of the inventive method where the disease or condition inthe mammal sought to be treated is a respiratory disease or conditionhaving as a symptom thereof abnormally high bronchial smooth muscle cellcontractility (i.e. excessive bronchoconstriction), the inventive methodcomprises administering an inventive composition to the mammal which iseffective at inhibiting the acetylcholine (AC) mediated contraction of abronchial smooth muscle cell or tissue. Inventive compositionscomprising the uPA kringle as an isolated kringle or as a part of theATF, tcuPA or the deletion mutant scuPA^(Δ136-143) are capable ofinhibiting the contractility of a bronchial smooth muscle cell ortissue, and thus are potent bronchodilators which can be used to treatsuch respiratory diseases or conditions. Examples of preferredrespiratory diseases and conditions in this embodiment include asthma,adult respiratory distress syndrome, primary pulmonary hypertension,microvascular thrombotic occlusion and disorders associated with chronicintrapulmonary fibrin formation.

In contrast, in embodiments of the inventive method where the disease orcondition in the mammal sought to be treated is characterized byabnormally low vascular smooth muscle cell or tissue contractility(e.g., hypotension) the uPA kringle is administered as part of aninventive composition capable of enhancing PE-induced contraction of amammalian vascular smooth muscle cell or tissue. For example, in theseembodiments, the uPA kringle is administered as an isolated kringle oras a part of a polypeptide of the invention such as ATF, tcuPA,scuPA^(Δ136-143), ATF+connecting peptide or uPA kringle+connectingpeptide.

In other embodiments, where the disease or condition of the mammalsought to be treated is characterized by abnormally high vascular smoothmuscle cell or tissue contractility (e.g., hypertension), the inventivemethod comprises administering to the mammal an inventive compositioncomprising the uPA growth factor domain as an isolated growth factordomain, or as a part of a polypeptide of the invention such as scuPA orΔkringle-scuPA or Δkringle-tcuPA, in an amount effective to inhibitPE-induced contraction of a vascular smooth muscle cell or tissue.

The invention also includes a method of identifying a compound which isan agonist or antagonist of one or more of the uPA kringle or a bindingprotein thereof, the uPA growth factor domain or a binding proteinthereof, and the connecting peptide or a binding protein thereof, uponthe contractility or angiogenic activity of a mammalian muscle orendothelial cell or tissue. The compound can be any type of compound,and includes by way of example and not by limitation, a drug, a peptide,a polypeptide, a protein, an oligonucleotide, a nucleic acid, a ligand,and an antibody.

The agonist can be any compound capable of enhancing, amplifying orpromoting the effect of the domain of uPA or binding protein thereofupon the contractility or angiogenic activity of a mammalian muscle orendothelial cell or tissue. The agonist can also be any compound whichis capable of enhancing, amplifying or promoting a transduction pathwayor degradation pathway of the domain of uPA or binding protein thereofThe antagonist can be any compound which is a competitive inhibitor orother inhibitor of the effect of the domain of uPA or binding proteinthereof upon the contractility or angiogenic activity of a mammalianmuscle or endothelial cell or tissue. The antagonist can also be anycompound which is a competitor or inhibitor of a transduction pathway ordegradation pathway of the domain of uPA or binding protein thereof. Thebinding protein can be any protein capable of specifically binding withone or more domains of uPA.

The method comprises assessing the contractility or angiogenic activityof a first mammalian muscle or endothelial cell and an otherwiseidentical second mammalian muscle or endothelial cell. The contractilityor angiogenic activity of the cells can be assessed by any method knownin the art or described herein for evaluating the contractility orangiogenic activity of a muscle or endothelial cell or tissue. Apreferred method for assessing contractility is described herein in theexperimental Examples.

The angiogenic activity of the cells and the effectiveness of a testcompound for modulating angiogenic activity can be assessed by examiningthe growth and migration of cultured endothelial cells or smooth musclecells in progressively less luxurious media and in response to knownchemotactic agents in vitro in the presence or absence of test compound.Cultures can be wounded, and the proliferation and capacity of cells torepair the wound in the presence or absence of test compound can beexamined by assays known in the art. In vitro assays can be used such asplacing growth factors in subcutaneous pockets of laboratory animals andmeasuring the ingrowth of vessels. One assay known in the art involvesthe use of chicken allantoic membranes (CAMs), where CAMs are implanted,and the vessel growth in response to locally infused agents forpromoting angiogenesis is measured. A test compound can also beadministered to an animal with a spontaneous or implanted tumor, and theangiogenic response of the tumor is evaluated by evaluating itsvasculature.

The method thereafter comprises providing to the first cell and thesecond cell a composition of the invention comprising one or more of theuPA kringle, the uPA growth factor domain and the connecting peptide, inan amount effective to modulate the contractility or angiogenic activityof a mammalian muscle or endothelial cell or tissue. The composition isprovided to the first and second cells by any method of providing acomposition to a cell described herein. The composition need not beprovided to the interior of the cells, but instead, where the first andsecond cells are in a mammal or in a tissue, the composition of theinvention need only be provided to the vascular system of the tissue ormammal.

The method also includes providing to the first cell a compoundsuspected to be an agonist or antagonist of one or more of the uPAkringle or a binding protein thereof, the uPA growth factor domain or abinding protein thereof, and the connecting peptide or a binding proteinthereof, upon the contractility or angiogenic activity of a mammalianmuscle or endothelial cell or tissue. The compound is provided to thefirst cell by any of the methods described herein or known in the artfor providing a composition or compound to a cell. The compound need notbe provided to the interior of the cell, since a binding protein of theuPA kringle, the uPA growth factor domain, and the connecting peptidecan be found either inside or outside of the cell (i.e., as a solubleprotein). The compound is provided to the first cell in an amountsuspected to be effective to modulate the effect of the composition ofthe invention upon the contractility or angiogenic activity of amammalian muscle or endothelial cell or tissue. The compound can beprovided to the first cell either simultaneous with, prior to, orsubsequent to providing the first cell the composition of the invention.

The method includes assessing the contractility or the angiogenicactivity of the first cell after providing to the first cell both thecomposition of the invention and the compound, and assessing thecontractility or the angiogenic of the second cell, which was notprovided the compound, after providing to the second cell thecomposition of the invention.

A preferred method for evaluating the contractility of a mammalian cellor tissue is described herein in the experimental Examples. Briefly, thecontractility of a cell and the effect upon the contractility of a cellcan be assessed by determining whether an increase or decrease isobserved in the EC₅₀ (50% effective concentration) for an inducingcompound which mediates contraction of the cell. For example, the EC₅₀can be evaluated for an inducing compound such as PE or AC to determinewhether the EC₅₀ changes in the first cell after administration of thecompound and the composition of the invention. In comparing any changein the EC₅₀ observed in the first cell with the EC₅₀ for PE in thesecond cell which is provided the composition of the invention but notthe compound, a decrease in the EC₅₀ of a cell for PE is an indicationof enhanced or disinhibited contractility in the cell, whereas anincrease in the EC₅₀ of a cell for PE is an indication of inhibitedcontractility in the cell. Such methods are described herein in theexperimental Examples, which describe methods for determining the EC₅₀for an inducing compound such as PE or AC in a muscle cell duringcontraction.

The method also comprises determining the effect upon the contractilityor angiogenic activity of the first cell by comparing the contractilityor angiogenic activity of the first cell before providing thecomposition of the invention with the contractility or angiogenicactivity of the first cell after providing both the compound and thecomposition of the invention. The effect upon the contractility orangiogenic activity of the second cell is also determined by comparingthe contractility or angiogenic activity of the second cell beforeproviding the composition of the invention with the contractility orangiogenic activity of the second cell after providing the compositionof the invention.

The method thereafter comprises comparing the effect upon contractilityor angiogenic activity observed in the first cell with the effect uponcontractility or angiogenic activity observed in the second cell. If theeffect of the composition of the invention upon contractility orangiogenic activity in the first cell is either increased or decreasedby at least about ten percent relative to the effect of the compositionof the invention upon contractility or angiogenic activity in the secondcell, then a compound is identified which is an agonist or antagonist ofone or more of the uPA kringle or a binding protein thereof, the uPAgrowth factor domain or a binding protein thereof, and the connectingpeptide or a binding protein thereof, upon the contractility orangiogenic activity of a mammalian muscle or endothelial cell or tissue.

The invention also includes a method of treating a disease or conditionin a mammal having as a symptom thereof one or more of abnormal musclecell or tissue contractility and abnormal angiogenic activity. Thedisease or condition can be any of the diseases or conditions describedherein. Preferably, the disease or condition is one which has as asymptom thereof abnormal vascular smooth muscle cell or tissuecontractility or abnormal bronchial smooth muscle cell or tissuecontractility.

The method comprises administering to the mammal an amount suspected tobe effective for modulating the contractility and/or angiogenic activityof a mammalian muscle or endothelial cell or tissue of an agonist orantagonist of one or more of the uPA kringle or a binding proteinthereof, the uPA growth factor domain or a binding protein thereof, andthe connecting peptide or a binding protein thereof. The agonist orantagonist can be administered to the mammal either alone or in the formof a pharmaceutical composition as described herein. The compound can beadministered by any route of administration described herein or known tothe skilled artisan for administering a compound to a mammal.

Preferably, the agonist or antagonist is administered to the mammal inan amount effective to modulate the effect of one or more of the uPAkringle or a binding protein thereof, the uPA growth factor domain or abinding protein thereof, and the connecting peptide or a binding proteinthereof upon the contractility of a muscle tissue in the mammal.

The method comprises providing the agonist or antagonist to a muscle orendothelial cell or tissue in the mammal having abnormal contractilityor abnormal angiogenic activity, or to a tissue or fluid of the mammalwhich is contiguous therewith. The abnormal contractility or angiogenicactivity of the muscle or endothelial cell or tissue can be eitherabnormally high or abnormally low contractility. The agonist orantagonist can be provided to the cell or tissue by any method describedherein for providing a compound to a cell directly, such as bycontacting the compound with the cell, or indirectly, such as throughthe action of any biological process. The agonist or antagonist need notbe provided to the interior of the cell, since a binding protein of theuPA kringle, a binding protein of the uPA growth factor domain, and abinding protein of the connecting peptide can be found either inside oroutside of the cell (i.e. as a soluble protein).

The method also comprises modulating the effect of one or more of theuPA kringle or a binding protein thereof, the uPA growth factor domainor a binding protein thereof, and the connecting peptide or a bindingprotein thereof, upon the muscle or endothelial cell or tissue havingabnormal contractility or abnormal angiogenic activity.

By modulating the effect of one or more of the uPA kringle or a bindingprotein thereof, the uPA growth factor domain or a binding proteinthereof, and the connecting peptide or a binding protein thereof uponthe contractility or angiogenic activity of the muscle or endothelialcell or tissue having abnormal contractility or abnormal angiogenicactivity, a disease or condition in the mammal having abnormal musclecell or tissue contractility or abnormal angiogenic activity as asymptom thereof is treated.

By way of example, and not by limitation, in one aspect of the method,the disease or condition treated is the vascular disease hypertension,which has as a symptom thereof abnormally high smooth muscle cellcontractility in a vascular smooth muscle cell or tissue. In thisembodiment, the mammal is administered an amount suspected to beeffective of one or more of an antagonist to the uPA kringle, anantagonist to a binding protein of the uPA kringle, an agonist of theuPA growth factor domain, an agonist of a binding protein of the uPAgrowth factor domain, an agonist of the connecting peptide, and anagonist of a binding protein of the connecting peptide. In thisembodiment, the vascular condition of hypertension in the mammal istreated by inhibiting the contractility enhancing effect of the uPAkringle or one of its binding proteins, or by enhancing the vasodilatingeffect of the growth factor domain and the connecting peptide and theirbinding proteins.

In another embodiment of the method, where the disease or conditiontreated in the mammal is a disease or condition having as a symptomthereof abnormally high bronchial smooth muscle cell or tissuecontractility (e.g., asthma), the method comprises administering to themammal an amount suspected to be effective of one or more of an agonistto the uPA kringle and an agonist to a binding protein of the uPAkringle. In this embodiment, the effect of the uPA kringle in naturallyoccurring tcuPA at inhibiting bronchial smooth muscle cell contractility(i.e. bronchodilation) is enhanced by the agonist in order to enhancebronchodilation in asthma. Preferably the agonist is administered in anamount effective to promote, increase or amplify the effect of the uPAkringle in naturally occurring tcuPA at inhibiting the contractility ofa bronchial smooth muscle tissue, thereby treating asthma in the mammal.

The invention also includes a method of determining whether a testprotein is a binding protein of one or more of the uPA kringle, the uPAgrowth factor domain and the connecting peptide. The method comprisesassessing the contractility modulating effect or the angiogenic activitymodulating effect of one or more of the uPA kringle, the uPA growthfactor receptor and the connecting peptide, in an amount effective tomodulate the contractility or angiogenic activity of a mammalian muscleor endothelial cell, upon a first cell or tissue, which either comprisesthe test protein or which is contiguous with a tissue or fluid of amammal which comprises the test protein. The test protein need not bepresent in the first cell or tissue, but can instead be a soluble orcirculating protein of a tissue or fluid which is contiguous with thefirst cell or tissue. For example, the test protein can be a knownsoluble protein of the bloodstream or lymphatic tissue of a mammal whichis capable of direct molecular signaling or signaling indirectly throughanother molecule to the first cell or tissue.

As with the test protein, the one or more of the uPA kringle, the uPAgrowth factor receptor and the connecting peptide can either be providedto the first cell or tissue, or to a tissue or fluid which is contiguouswith the first cell or tissue, such as a blood or lymph cell or tissue.

The contractility modulating effect or angiogenic activity modulatingeffect of one or more of the uPA kringle, the uPA growth factor receptorand the connecting peptide, in an amount effective to modulate thecontractility or angiogenic activity of a mammalian muscle orendothelial cell, is also assessed in a second, otherwise identical,cell or tissue which does not comprise the test protein and which is notcontiguous with a tissue or fluid which comprises the test protein. Theone or more of the uPA kringle, the uPA growth factor receptor and theconnecting peptide can either be provided to the second cell or tissue,or to a tissue or fluid which is contiguous with the second cell ortissue, such as a blood or lymph cell or tissue.

The contractility modulating effect or the angiogenic activitymodulating effect in the first cell or tissue is then compared with thecontractility modulating effect or the angiogenic activity modulatingeffect in the second cell or tissue. If the degree of modulation ofcontractility or angiogenic activity by one or more of the uPA kringle,the uPA growth factor receptor and the connecting peptide is greater inthe first cell or tissue relative to the second cell or tissue, then thetest protein is a binding protein of one or more of the uPA kringle, theuPA growth factor receptor and the connecting peptide. The contractilityor angiogenic activity of the cells and the contractility modulatingeffect or angiogenic activity modulating effect of one or more of theuPA kringle, the uPA growth factor receptor and the connecting peptideupon the cells can be assessed using any method known in the art ordescribed herein.

In one embodiment, the contractility modulating effect or the angiogenicactivity modulating effect of one or more of an isolated kringle, theATF, tcuPA, and a peptide or functional element of the uPA kringle isassessed and compared in a first and second cell or tissue. In thisembodiment, if a greater increase in contractility or a greater decreasein angiogenic activity is observed in the first cell or tissue relativeto the second cell or tissue, then the test protein is a binding proteinof one or more of an isolated kringle, the ATF, tcuPA and a peptide orfunctional element of the uPA kringle.

When present inside the first or second cells, the test protein and/orthe one or more of the uPA kringle, the uPA growth factor receptor andthe connecting peptide can be present, by way of example and not bylimitation, either naturally or as an expressed recombinant polypeptide,or by providing the test protein and/or one or more of the uPA kringle,the uPA growth factor receptor and the connecting peptide to the cellsas a component of any of the compositions of the invention describedherein using any of the methods described herein for providing acomposition or compound to a cell.

The test protein and/or one or more of the uPA kringle, the uPA growthfactor receptor and the connecting peptide can also be present in thecells as a naturally occurring protein or as an expressed recombinantpolypeptide resulting from the transfection of the cell with any of thevectors described herein comprising a recombinant polynucleotide whichencodes the test protein and/or one or more of the uPA kringle, the uPAgrowth factor receptor and the connecting peptide using any method knownin the art or described herein or known in the art to transfect amammalian cell. The recombinant polynucleotide encoding such arecombinant polypeptide test protein can be prepared by any method knownin the art or described herein.

In addition to the method described above, a binding protein of one ormore of the uPA kringle, the uPA growth factor receptor and theconnecting peptide can be identified using conventional methods known inthe art. For example, immunological methods can be used to identify abinding complex of one or more of the uPA kringle, the uPA growth factorreceptor and the connecting peptide with a test protein, the specificityof binding and the affinity of the binding complex being an indicationthat the test protein is a binding protein of one or more of the uPAkringle, the uPA growth factor receptor and the connecting peptide. Inaddition, the identification of an effect upon any signal transductionevent (e.g. calcium release) associated with one or more of the uPAkringle, the uPA growth factor receptor and the connecting peptide canbe used to identify a test protein as a binding protein thereof inassays known in the art for assessing signal transduction. Test proteinsidentified as having an effect upon signal transduction by one or moreof the uPA kringle, the uPA growth factor receptor and the connectingpeptide are thus identified as binding proteins thereof.

The invention also includes a method of identifying a functional elementof one or more of the uPA kringle, the uPA growth factor receptor andthe connecting peptide. The functional element participates in themodulation of contractility or angiogenic activity of a mammalian muscleor endothelial cell or tissue. The method comprises preparing one ormore mutant polypeptides which lack a portion of the amino acid sequenceof one or more of the uPA kringle, the uPA growth factor receptor andthe connecting peptide. These mutant polypeptides can be prepared by anymethod known in the art for preparing deletion mutant proteins orpolypeptides. Examples of such methods are described herein in theexperimental Examples. The portions of the amino acid sequence which areto be deleted can be determined either randomly or in a directionalmanner proceeding from either the N-terminal or C-terminal end of thepolypeptide and proceeding in a single direction.

The method also comprises assessing the ability of each deletion mutantso prepared to modulate the contractility or to modulate the angiogenicactivity of a mammalian muscle or endothelial cell or tissue onceprovided to the cell or tissue, or to a tissue or fluid which iscontiguous with the cell or tissue, as discussed herein. If provided tothe cell or tissue; the mutant polypeptide can be provided using anymethod described herein for providing a composition to a cell or tissue.The modulation of the contractility or angiogenic activity can be eitheran enhancement, a disinhibition or an inhibition of the contractility orangiogenic activity of any of the muscle or endothelial cell or tissuetypes described herein. The contraction of the cell can either bemediated or not mediated by any of the inducing compounds describedherein. The modulation of contractility or angiogenic activity in amuscle or endothelial cell which is provided a mutant polypeptide can beassessed by any of the methods described herein or known in the art. Forexample, a decrease or increase in the EC₅₀ of an inducing compoundduring contraction of the muscle cell can be used to assess the abilityof the mutant polypeptide to modulate the contractility of the cell ortissue, once provided to the cell or tissue, or to a tissue or fluidwhich is contiguous therewith.

The method also includes identifying, from the assessment describedabove, a mutant polypeptide which is not able to modulate thecontractility or which is not able to modulate the angiogenic activityof a mammalian muscle or endothelial cell. Based on the identificationof a specific mutant polypeptide, the corresponding deleted portion ofthe amino acid sequence of one or more of the uPA kringle, the uPAgrowth factor receptor and the connecting peptide which participates inthe modulation of contractility or angiogenic activity of a muscle orendothelial cell is determined. Thus, a functional element of one ormore of the uPA kringle, the uPA growth factor receptor and theconnecting peptide is identified.

In addition to the method discussed above, other methods known in theart for identifying a functional element of a polypeptide can be used.For example, anti-peptide antibodies, deletion/substitution mutants,chimeric polypeptides and other peptide inhibition methods can be usedto identify a functional element of one or more of the uPA kringle, theuPA growth factor receptor and the connecting peptide by methods knownin the art.

The invention also includes a method of treating a vascular disease orcondition in a mammal having as a symptom thereof abnormally high fibrinclot formation. The method comprises administering to the mammal acomposition comprising one or more of Δkringle-scuPA, Δkringle-tcuPA, anantagonist of the uPA kringle and an antagonist of a binding protein ofthe uPA kringle in an amount effective to inhibit the contractility of amammalian vascular smooth muscle cell or tissue. The polypeptidesΔkringle-scuPA and Δkringle-tcuPA are deletion mutants of theurokinase-type plasminogen activator protein lacking the uPA kringle,which share at least about 75% homology with the polypeptidecorresponding to SEQ ID NO:7 (FIG. 1G). Preferably, Δkringle-scuPA andΔkringle-tcuPA are about 80% homologous, more preferably about 85%homologous, even more preferably 90% homologous, yet more preferably 95%homologous, and most preferably about 99% homologous to the polypeptidecorresponding to SEQ ID NO:7. Even more preferably, Δkringle-scuPA andΔkringle-tcuPA are each the polypeptide corresponding to SEQ ID NO:7.The antagonist of the uPA kringle and of the uPA kringle binding proteincan be any of those described herein.

The method also comprises providing the composition to an affected cellor tissue of the cardiovascular system of the mammal which has or isprone to develop excessive fibrin clot formation, or to a tissue orfluid of the mammal which is contiguous therewith. The composition canbe provided using any method described herein for providing to a cell ortissue a composition of the invention. A sufficient period of time ispermitted for the uPA protease domain component of the composition, ifpresent, to initiate the breakdown of fibrinogen and the process offibrin clot lysis and for the composition to cause vasodilation in theaffected area by inhibiting the contractility of the vascular smoothmuscle cell or tissue in the affected area. Thus, the method results inthe promotion of both fibrin clot lysis and vasodilation in the affectedarea of the vasculature of the mammal, thereby treating the vasculardisease or condition.

The method can be used to promote both clot lysis and vasodilation inany vascular tissue in a mammal. The clot can be of any natural origin,such as from any of the diseases or conditions described herein, or ofany artificially induced origin, for example, post-traumatic clotting,such as after vascular surgery, stenting or angioplasty. Conventionaltherapy involves the use of tcuPA to promote clot lysis. However, usingtcuPA has the disadvantage of also causing vasoconstriction in theaffected area, thus partially defeating the purpose of clot lysis (i.e.to increase blood flow through a vessel). Thus, using Δkringle-tcuPA topromote clot lysis instead of tcuPA has the advantage of overcoming thedeleterious vasoconstrictive properties of tcuPA which arise from thecontractility enhancing effect of the uPA kringle, while retaining theclot lysis activity of the uPA protease domain. Alternatively,Δkringle-scuPA can be administered to the mammal along with arecombinant version of soluble uPA receptor (suPAR). In this embodiment,the single chain molecule undergoes a conformational change whichinvests it with clot lysis activity and resistance to its plasmainhibitors. Thus, both Δkringle-scuPA and Δkringle-tcuPA are useful inthe inventive method of promoting both fibrin clot lysis andvasodilation in an affected vascular tissue of a mammal.

The invention also includes a kit for treating a disease or condition ina mammal, the disease or condition having as a symptom thereof one ormore of abnormal muscle cell or tissue contractility and abnormalangiogenic activity. The kit comprises a composition of the invention inan amount effective to modulate one or more of the contractility and theangiogenic activity of a mammalian muscle or endothelial cell or tissue.The kit also comprises an instructional material which describesadministering the composition to a muscle or endothelial cell or tissueof a mammal having abnormal contractility and/or abnormal angiogenicactivity.

In another embodiment, this kit comprises a (preferably sterile) solventsuitable for dissolving or suspending the composition of the inventionprior to administering the composition to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionin the kit for effecting treatment of the various diseases or conditionsrecited herein. Optionally, or alternatively, the instructional materialmay describe one or more methods of treating a disease or conditiondescribed herein. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the peptideof the invention or be shipped together with a container which containsthe peptide. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the compound be used cooperatively by the recipient.

The invention is now described with reference to the following Examples.The Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLE 1

The objective of the experiments described in this Example was toevaluate the ability of single chain (scuPA) and two chain (tcuPA)urokinase, as well as isolated fragments of urokinase-type plasminogenactivator (uPA), to modulate vascular smooth muscle cell (SMC)contractility. Single chain uPA (scuPA) was found to inhibitphenylepherine (PE)-induced contraction of rat aortic rings, whereas twochain uPA (tcuPA) was found to exert the opposite effect. Twoindependent epitopes mediating these opposing activities wereidentified. A single chain uPA deletion mutant lacking amino acids136-143 (the connecting peptide domain) did not induce vasorelaxation. Asecond epitope within the uPA kringle enhanced PE-inducedvasoconstriction. This epitope was exposed when single chain uPA wasconverted to a two-chain molecule by plasmin. The isolated uPA kringleaugmented vasoconstriction whereas the uPA variant lacking the kringlehad no procontractile activity. These studies reveal previouslyundescribed vasoactive domains within urokinase and itsnaturally-derived fragments. The materials and methods used in theexperiments discussed in this Example are described below.

Evaluation of Contractility

Male Sprague Dawley rats (250-275 g) were sacrificed by exsanguinationand thoracic aortae were removed with care to avoid damage to theendothelium, dissected free of fat and connective tissue, and cut intotransverse rings, each 5 mm in length (Kyong et al., 1992, Br. J.Pharmacol. 107:983-990; Yohtaro et al., 1987, Eur. J. Pharmacol.131:75-78; Chang et al., 1992, Br. J. Pharnacol. 107:983-990; Oyama etal., 1986, Eur. J. Pharmacol. 132:75-78). The tissues were maintained inan oxygenated (95% O₂; 5% CO₂) solution of Krebs-Henseliet (KH) buffer(144 millimolar NaCl, 5.9 millimolar KCl, 1.6 millimolar CaCl₂, 1.2millimolar MgSO₄, 1.2 millimolar KH₂PO₄, 25 millimolar NaHCO₃, and 11.1millimolar D-glucose). The aortic rings were mounted to record isometrictension in a 10 milliliter bath containing KH solution under continuousaeration. The aortic rings were equilibrated for 1.5 hours at 37° C. andmaintained under a resting tension of 2 g throughout the experiment.Each aortic ring was then contracted by adding phenylepherine (PE) instepwise increments raising the concentration of PE from 10⁻¹⁰ molar to10⁻⁵ molar. In other experiments, various concentrations of scuPA, tcuPAor uPA fragments were added 15 minutes prior to adding PE. In everyexperiment, aortic rings exposed to KH buffer alone were analyzed inparallel as controls. Isometric tension was evaluated using a forcedisplacement transducer and recorded online using a computerized system(ExperimentiaAE, Budapest, Hungary).

In some experiments two chain urokinase (tcuPA; a gift of AmericanDiagnostica, Greenwich Conn.) was studied. tcuPA was documented as freeof the isolated amino terminal fragment of uPA on native gels. In otherexperiments, scuPA or scuPA variants (see below) (20 micromolar each)were incubated with plasmin (0.1 micromolar) for 30 minutes at 370° C.to generate tcuPA. The mixture was added to soluble recombinant humanurokinase receptor (suPAR) (see below) bound to CnBr-activated Sepharose(Sigma, St. Louis, Mo.) for one hour at 4° C. (Higazi et al., 1998,Blood 92:2075-2083). The Sepharose beads were washed extensively and thetcuPA was released by adding glycine buffer, pH 3.0. The activity oftcuPA was assessed using the chromogenic substrate S-2444 (Higazi etal., 1996, Thromb. Res. 84:243-252). The completeness of the conversionof scuPA to tcuPA was verified using sodium dodecylsulfate-polyacrylimide gel electrophoresis (SDS-PAGE) under reducingconditions. The preparation was found to be free of plasmin as judged bycleavage of its chromogenic substrate S-2251 (Chromogenics).

Preparation of Deletion Mutants scuPA^(Δ136-143) and Δkringle-scuPA

A plasmid encoding scuPA^(Δ136-143) was generated using a two steppolymerase chain reaction. The cDNA encoding full length scuPA inpcUK176 served as the template. The primers 5′-CGCGGATCCAGCAATGAAC-3′(SEQ ID NO:19) and 5′-TGGCCACACTGAAATTTTAATTTTCCATCTGCGCAGTCAT-3′ (SEQID NO:20) were used to generate the 438 bp 5′-fragment pUN121 (Axelrod,1989, Molecular and Cellular Biology 9:2133-2141), while5′-TTAAAATTTCAGTGTGGCCA (SEQ ID NO:21) and 5′-CCAAGCT CGAGGTGCCCG (SEQID NO:22) were used to generate the 834 bp 3′ fragment. After completionof the second PCR step, the final product was digested using BamHI andXhoI and directionally subcloned into pMT/Bip (Invitrogen, Carlsbad,Calif.) to yield scuPA^(Δ136-143)/pMT/Bip. The recombinant protein wasexpressed using the Drosophila Expression System (Invitrogen) inSchneider S2 cells according to the manufacturer's recommendations.scuPA^(Δ136-143)/pMT/Bip was purified from S2 medium by affinitychromatography using immobilized rabbit polyclonal anti-human uPARantibody (Immunopure Protein G IgG Plus Orientation Kit, Pierce ChemicalCo., Rockford, Ill.). scuPA lacking residues 47-135 (Δkringle-scuPA) wasgenerated by a two step PCR procedure in the same manner. The fragmentwas digested using Bgl II and Xho I, subcloned into pMT/BiP (Invitrogen)and expressed in Schneider 2 cells. Δkringle-scuPA was purified from thecell supernatant using SP-Sephadex (Pharmacia, Piscataway, N.J.) andHPLC chromatography. The deletion mutant polypeptide was characterizedby SDS-PAGE under reducing and non-reducing conditions followed byWestern blot analysis using anti-urokinase antibody.

Purification of uPA Kringle

A sample of tcuPA (7.5 mgs/mL) was dialyzed against 0.1 molar sodiumphosphate containing 0.6 molar sodium chloride, pH 7.8. Plasmin wasadded to the dialyzed sample at a final concentration of 1 micromolarand the mixture was incubated at 370° C. for 48 hrs. Pefablock was addedat a concentration of 1 micromolar to quench the reaction and the uPAkringle was purified using reverse phase HPLC (RP-HPLC) on a C₈ column.N-terminal sequencing analysis of the purified uPA kringle confirmed theN-terminus as starting with Ser⁴⁷ of the mature uPA sequence and themass of the uPA kringle was determined using Matrix Assisted LaserDesorption Ionisation—Time of Flight (MALDI-TOF) mass spectrometry to beconsistent with a composition corresponding to amino acids 47-135 ofuPA, having a molecular weight of 10138 Da. The uPA kringle was found tobe greater than 95% pure using SDS-PAGE and greater than 99% pure usinganalytical C₈ RP-HPLC.

Preparation of Recombinant, Soluble Urokinase Receptor (suPAR)

A cDNA encoding full length uPAR in uPAR/pGEM was used as a template ina PCR reaction to introduce a stop codon after amino acid residue 277and to introduce restriction enzyme sites (Higazi et al., 1996,Biochemistry 35:6884-6890). The PCR product was subcloned into pMT/Bipat the Bgl II and XhoI sites. The complete sequence was confirmed usingautomated fluorescence-based sequence analysis. suPAR was expressedusing the Drosophila Expression System in Schneider S2 cells asdescribed above and purified using affinity chromatography onscuPA-Sepharose (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380).

Assessment of Binding of scuPA^(Δ136-143) and Kringle-scuPA to theUrokinase Receptor

The binding of wild type scuPA, scuPA^(Δ136-143), and Δkringle-scuPA tothe urokinase receptor (uPAR) was assessed using a BIA 3000 opticalBiosensor (Biacore, AB, Sweden) (Higazi et al., 1996, Biochemistry35:6884-6890). This method detects binding interactions in real time bymeasuring changes in the refractive index (RI) at a biospecific surface,enabling the calculation of association and dissociation rate constants.For these studies, suPAR was coupled to a CM5-research grade sensor chipflow cell (Biacore, AB, Sweden) via standard amine coupling proceduresusing N-hydroxysuccinimide/N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimine hydrochloride (Pierce, Rockford, Ill.) at a level of500 response units (RU) (Johnsson et al., 1995, J. Mol. Recognit.8:125-131). The sensor surface was coated using 10 microgram permilliliter solution of suPAR in 10 micromolar sodium acetate buffer, pH5.0. After immobilization, unreacted groups were blocked with 1 molarethanolamine, pH 8.5. A second flow cell which was similarly activatedand blocked without immobilization of protein served as a control. Allbinding reactions were performed in phosphate buffered saline, pH 7.4,containing 0.005% TWEEN-20. The binding of scuPA (both wild type andscuPA^(Δ136-143)) was measured at 25° C. at a flow rate of 60microliters per minute for 2 minutes, followed by 2 minutes ofdissociation. The bulk shift due to changes in RI was measured using theblank surface, and this value was subtracted from the binding signal ateach condition to correct for non-specific signals. Binding surfaceswere regenerated using two 18 second pulses of 1 molar NaCl, pH 3.5,followed by an injection of binding buffer for 1 min. to remove thishigh salt solution. All injections were performed in a random fashionusing the RANDOM command in the automated method. The binding ofurokinase was measured over a range of concentrations from 12.5nanomolar to 0.2 nanomolar. Data were collected at 2.5 Hz, and fit to a1:1 Langmuir reaction mechanism using global analysis in the BIAevaluation 3.0 software (Biacore, AB, Sweden). Secondary plots of thedata [ln(|dR/dt|) vs time] were also performed to unmask anycontribution from mass transport to the kinetic data.

The results of the experiments presented in this Example are nowdescribed.

The effect of scuPA and tcuPA upon the phenylepherine (PE) inducedcontraction of isolated rat aortic rings was studied first. Both ofthese forms of urokinase contain an identical amino acid sequence, butthe molecules differ in their intrinsic enzymatic activity andsusceptibility to plasminogen activator (PA) inhibitors. As indicated inFIG. 2, scuPA inhibited PE induced contraction of the aorta, increasingthe EC₅₀ from 29 nanomolar to 182 nanomolar. scuPA was converted totcuPA as described in the “Materials and Methods” section above byadding plasmin, which cleaves Lys¹⁵⁸-Ile¹⁵⁹, generating a moleculecomposed of two chains that are linked by a single disulfide bond. Theresulting tcuPA was affinity purified using immobilized solubleurokinase receptor (uPAR) or benzamidine to remove plasmin. In directcontrast to scuPA, tcuPA was found to enhance PE-induced aorticcontraction, decreasing the EC₅₀ of PE from 29 nanomolar to 4.47nanomolar as shown in FIG. 2.

While not wishing to be bound by any particular theory, it is postulatedthat single chain and two chain full-length uPA have opposing effects onvascular contractility because they differ in which biologically activeepitopes are exposed and/or because the intramolecular interactionsamong the domains differ. In order to distinguish between thesepossibilities, experiments were conducted using isolated fragments ofurokinase.

LMW-uPA, which lacks the amino-terminal fragment and is catalyticallyactive in tcuPA, but nascent in scuPA, exhibited no effect uponPE-induced contraction of aortic rings at concentration as high as 20micromolar. Therefore, the amino terminal fragment of urokinase (ATF,amino acids 1-135), the portion of the urokinase molecule which binds touPAR was studied. First, the effect of soluble uPAR (suPAR) uponuPA-mediated vasoconstriction was examined. Binding with suPARcompletely abolished the contractility modulating effects of scuPA andtcuPA as evidenced by a return of the EC₅₀ to 29 nanomolar. This outcomesuggested that uPA-induced aortic contraction was mediated by ATF or aportion of uPA in close enough spatial proximity that it is no longeravailable when the molecule is bound to its receptor in solution.Isolated ATF was itself found to enhance contractility, as evidenced bythe decrease in the EC₅₀ for PE to 0.71 nanomolar shown in FIG. 3.Indeed, isolated ATF was found to be 6-fold more potent than tcuPA atenhancing vascular smooth muscle tissue contractility (compare FIGS. 2and 3) and its activity was abolished by suPAR as well (FIG. 3).

The aminoterminal fragment of urokinase (ATF) is composed of twosubdomains, the growth factor domain (GFD) and the kringle. The GFD isknown to mediate the binding of uPA to its receptor. The function of theuPA kringle has heretofore been unknown. In order to determine whichportion of ATF was responsible for the contractility enhancing effect oftcuPA and ATF, the effect of each isolated fragment on PE-inducedvasoconstriction was assessed. As indicated in FIG. 4, the uPA kringledid not induce vasoconstriction directly, but instead markedly enhancedthe effect of phenylepherine. The addition of the uPA kringle at aconcentration of 1 nanomolar decreased the EC₅₀ from 29 nanomolar to 0.1nanomolar, whereas GFD added at a concentration of 10 nanomolar did notenhance contractility.

Taken together, these findings indicate that the contractility enhancingeffect of tcuPA is mediated by the uPA kringle, an effect which may beoverridden in scuPA by a signal involving an adjacent domain termed the“connecting peptide” of urokinase. Alternatively, the pro-contractileepitope of urokinase may be exposed in tcuPA but not in scuPA. Todistinguish between these possibilities, a variant of scuPA was preparedwhich lacked the connecting peptide (scuPA^(Δ136-143)), and thevasoactive properties of this variant before and after its conversion toa two-chain molecule were evaluated.

The urokinase variant scuPA^(Δ136-143) was found to exhibit no effectupon PE-induced aortic contraction, as indicated in FIG. 5A. This resultdemonstrates that the connecting peptide is required to inhibitcontraction and that the contractility enhancing elements in ATF are notactive in scuPA. Experiments were then addressed at whether the lack ofactivity of scuPA^(Δ136-143) was due to a loss of affinity for uPAR. Thebinding kinetics of wild type scuPA and scuPA^(Δ136-143) to suPARimmobilized on a CM5 sensor chip were measured using a BIA 3000 opticalbiosensor (see FIG. 6). The binding was found to be kinetically driven,as examination of secondary plots failed to reveal any limitationresulting from mass transport. The variant scuPA^(Δ136-143) bound tosuPAR with only a small reduction in Kd compared with wild type scuPA(See Table 1), a difference that cannot account for its complete loss ofvascular reactivity. Furthermore, scuPA^(Δ136-143) expressed the sameplasminogen activator activity as wild type scuPA in the presence ofsuPAR and after cleavage by plasmin to a two-chain molecule (Higazi etal., 1995, J. Biol. Chem. 270:17375-17380; Higazi et al., 1998, Blood92:2075-2083). In addition, the EC₅₀ for PE-induced contraction in thepresence of tcuPA^(Δ136-143) and wild type tcuPA were almost identical,as indicated in FIGS. 2 and 5A.

Taken together, this series of experiments suggests that the epitope(s)in the uPA kringle which modulate vascular smooth muscle cellcontractility are exposed only when scuPA is converted to tcuPA.

A similar experimental approach was used to determine whether thecontractility enhancing effect of tcuPA is due to the overridinginfluence of the uPA kringle above that of the connecting peptide, inthe full urokinase molecule, or whether there was a difference in theexposure of two independently functioning domains. In order todistinguish between these possibilities, the effect of a uPA variantlacking the kringle (Δkringle-uPA) upon PE-induced vasoconstriction wasassessed.

The kinetics of binding of wild type scuPA (WT), scuPA^(Δ136-143) andΔkringle-scuPA to immobilized suPAR were measured on the BIA3000 asdescribed in the text. Data from these experiments (sensorgrams shown inFIG. 5) were fit to a 1:1 Langmuir reaction mechanism using globalanalysis. The equilibrium dissociation constant (Kd) was calculated fromthe ratio of the kinetic constants (Kd=kd/ka). Table 1 shows theresulting association (ka) and dissociation (kd) rate constants.

TABLE 1 Analyte ka (M⁻¹s⁻¹) kd (s⁻¹) Kd (M) WT-scuPA 6.3 × 10⁶ 2.1 ×10⁻³ 3.3 × 10⁻¹⁰ ScuPA^(Δ136-143) 3.3 × 10⁶ 3.8 × 10⁻³ 1.1 × 10⁻⁹Δkringle-scuPA 3.8 × 10⁶ 2.2 × 10⁻³ 0.6 × 10⁻¹⁰

The results of these experiments indicated that Δkringle-scuPA and WTscuPA bound to suPAR with almost the same Kd (Table 1). The variantΔkringle-scuPA also inhibited PE-induced vasoconstriction to the sameextent as WT scuPA, as indicated in FIGS. 1 and 5B. However, tcuPAlacking the kringle was unable to enhance PE-induced vascular smoothmuscle cell contractility.

Summary

The results of the experiments described above help to reconcile some ofthe discrepant observations in urokinase-mediated signal transduction byproviding novel insights into the nature of intramolecular interactionswithin the urokinase molecule itself. The finding that single-chainurokinase and two-chain urokinase exert opposing effects upon smoothmuscle cell contractility provides not only the first described effectof this plasminogen activator upon contractility, but also providescompelling evidence for the existence of more than one functionallyactive site within the molecule.

One epitope, located within the connecting peptide (amino acids136-143), inhibits vasoconstriction induced by phenylepherine andendothelin by blocking the release of calcium from intracellular storesas well as its entry from the extracellular space. The effect of theconnecting peptide upon vasorelaxation is sequence specific and requiresboth Ser¹³⁸ and E¹⁴³. Further support for the concept that theconnecting peptide functions as a biologically active unit derives fromits capacity to inhibit cell migration and tumor cell invasiveness.

A second epitope, within the uPA kringle, enhances PE inducedvasoconstriction. This contractility enhancing epitope and the epitopewithin the connecting peptide which inhibits contractility are notexposed simultaneously in either single-chain or two-chain uPA. Only theconnecting peptide is exposed in scuPA. This conclusion is establishedby several findings. First, a variant of scuPA lacking only these eightamino acids neither causes vasorelaxation nor vasoconstriction. On theother hand, the epitope within the uPA kringle is exposed only in tcuPA.This conclusion is based on two observations: deleting the uPA kringledid not inhibit the effect of scuPA on vasorelaxation, but the deletionabolished its contractility enhancing activity as a two-chain molecule.Second, plasmin-mediated activation of scuPA exposes a contractilityenhancing epitope within the amino terminal fragment co-incident withthe loss of the inhibitory epitope within the connecting peptide. Thisconclusion derives from the observation that cleavage of the scuPAvariant lacking the connecting peptide by plasmin generates a two chainvariant molecule possessing the same pro-contractile potency as wildtype tcuPA but does not inhibit PE-induced vasoconstriction as a singlechain molecule.

Thus, the results of the experiments described above provide evidencethat a polypeptide comprising one or more domains of uPA, selected fromthe uPA kringle, the growth factor domain and the connecting peptide, iseffective at modulating the contractility of a mammalian muscle cell ortissue, and can be used advantageously as part of a composition to beadministered to a mammal for the treatment of a disease or conditionhaving as a symptom thereof abnormal muscle cell or tissuecontractility.

EXAMPLE 2

The objective of the experiments described in this Example was toevaluate the ability of the uPA kringle to modulate bronchial smoothmuscle cell or tissue contractility. The effect of the uPA kringle uponacetylcholine induced contraction of isolated rat tracheal rings wasevaluated. Isolated rat tracheal rings were either pre-incubated withmedia containing 10 nanomolar or 100 nanomolar uPA kringle or withcontrol media lacking the uPA kringle. Varying concentrations ofacetylcholine (AC) were then added as an inducing compound to inducecontraction and the tension in the rings upon AC-induced contraction wasmeasured. FIG. 8 depicts the results of these experiments, withcontractility being shown as a percent of the contraction of thecontrol. The mean i standard deviation of three independent experimentsis shown.

The results of these experiments indicate that the uPA kringle inhibitsthe contractility of rat tracheal tissue in a dose-dependent manner, asindicated by the increase in EC₅₀ for acetylcholine-induced contractionshown in FIG. 8 for tracheal rings pre-treated with either 10 nanomolaror 100 nanomolar uPA kringle. Thus, compositions comprising the uPAkringle can be used to treat respiratory diseases and conditions havingas a symptom thereof abnormally high bronchial smooth muscle cell ortissue contractility, such as asthma, adult respiratory distresssyndrome, primary pulmonary hypertension and chronic intrapulmonaryfibrosis.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

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Cys Leu Pro Trp Asn Ser Ala Thr Val Leu 65 70 75 80 Gln Gln ThrTyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu 85 90 95 Gly Lys HisAsn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp 100 105 110 Cys TyrVal Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val 115 120 125 HisAsp Cys Ala Asp Gly Lys Lys Pro Ser Ser Pro Pro Glu Glu Leu 130 135 140Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile 145 150155 160 Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile165 170 175 Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly GlySer 180 185 190 Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His Cys PheIle Asp 195 200 205 Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly ArgSer Arg Leu 210 215 220 Asn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu ValGlu Asn Leu Ile 225 230 235 240 Leu His Lys Asp Tyr Ser Ala Asp Thr LeuAla His His Asn Asp Ile 245 250 255 Ala Leu Leu Lys Ile Arg Ser Lys GluGly Arg Cys Ala Gln Pro Ser 260 265 270 Arg Thr Ile Gln Thr Ile Cys LeuPro Ser Met Tyr Asn Asp Pro Gln 275 280 285 Phe Gly Thr Ser Cys Glu IleThr Gly Phe Gly Lys Glu Asn Ser Thr 290 295 300 Asp Tyr Leu Tyr Pro GluGln Leu Lys Met Thr Val Val Lys Leu Ile 305 310 315 320 Ser His Arg GluCys Gln Gln Pro His Tyr Tyr Gly Ser Glu Val Thr 325 330 335 Thr Lys MetLeu Cys Ala Ala Asp Pro Gln Trp Lys Thr Asp Ser Cys 340 345 350 Gln GlyAsp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg Met 355 360 365 ThrLeu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp 370 375 380Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile Arg 385 390395 400 Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 405 410 4 135 PRTHomo sapiens 4 Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys LeuAsn Gly 1 5 10 15 Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile HisTrp Cys Asn 20 25 30 Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile AspLys Ser Lys 35 40 45 Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly LysAla Ser Thr 50 55 60 Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser AlaThr Val Leu 65 70 75 80 Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala LeuGln Leu Gly Leu 85 90 95 Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn ArgArg Arg Pro Trp 100 105 110 Cys Tyr Val Gln Val Gly Leu Lys Pro Leu ValGln Glu Cys Met Val 115 120 125 His Asp Cys Ala Asp Gly Lys 130 135 5276 PRT Homo sapiens 5 Lys Pro Ser Ser Pro Pro Glu Glu Leu Lys Phe GlnCys Gly Gln Lys 1 5 10 15 Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly GlyGlu Phe Thr Thr Ile 20 25 30 Glu Asn Gln Pro Trp Phe Ala Ala Ile Tyr ArgArg His Arg Gly Gly 35 40 45 Ser Val Thr Tyr Val Cys Gly Gly Ser Leu IleSer Pro Cys Trp Val 50 55 60 Ile Ser Ala Thr His Cys Phe Ile Asp Tyr ProLys Lys Glu Asp Tyr 65 70 75 80 Ile Val Tyr Leu Gly Arg Ser Arg Leu AsnSer Asn Thr Gln Gly Glu 85 90 95 Met Lys Phe Glu Val Glu Asn Leu Ile LeuHis Lys Asp Tyr Ser Ala 100 105 110 Asp Thr Leu Ala His His Asn Asp IleAla Leu Leu Lys Ile Arg Ser 115 120 125 Lys Glu Gly Arg Cys Ala Gln ProSer Arg Thr Ile Gln Thr Ile Cys 130 135 140 Leu Pro Ser Met Tyr Asn AspPro Gln Phe Gly Thr Ser Cys Glu Ile 145 150 155 160 Thr Gly Phe Gly LysGlu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln 165 170 175 Leu Lys Met ThrVal Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln 180 185 190 Pro His TyrTyr Gly Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala 195 200 205 Asp ProGln Trp Lys Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro 210 215 220 LeuVal Cys Ser Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser 225 230 235240 Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg 245250 255 Val Ser His Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn260 265 270 Gly Leu Ala Leu 275 6 403 PRT Homo sapiens 6 Ser Asn Glu LeuHis Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly 1 5 10 15 Gly Thr CysVal Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn 20 25 30 Cys Pro LysLys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys 35 40 45 Thr Cys TyrGlu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr 50 55 60 Asp Thr MetGly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu 65 70 75 80 Gln GlnThr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu 85 90 95 Gly LysHis Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp 100 105 110 CysTyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val 115 120 125His Asp Cys Ala Asp Gly Lys Leu Lys Phe Gln Cys Gly Gln Lys Thr 130 135140 Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu 145150 155 160 Asn Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg Gly GlySer 165 170 175 Val Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys TrpVal Ile 180 185 190 Ser Ala Thr His Cys Phe Ile Asp Tyr Pro Lys Lys GluAsp Tyr Ile 195 200 205 Val Tyr Leu Gly Arg Ser Arg Leu Asn Ser Asn ThrGln Gly Glu Met 210 215 220 Lys Phe Glu Val Glu Asn Leu Ile Leu His LysAsp Tyr Ser Ala Asp 225 230 235 240 Thr Leu Ala His His Asn Asp Ile AlaLeu Leu Lys Ile Arg Ser Lys 245 250 255 Glu Gly Arg Cys Ala Gln Pro SerArg Thr Ile Gln Thr Ile Cys Leu 260 265 270 Pro Ser Met Tyr Asn Asp ProGln Phe Gly Thr Ser Cys Glu Ile Thr 275 280 285 Gly Phe Gly Lys Glu AsnSer Thr Asp Tyr Leu Tyr Pro Glu Gln Leu 290 295 300 Lys Met Thr Val ValLys Leu Ile Ser His Arg Glu Cys Gln Gln Pro 305 310 315 320 His Tyr TyrGly Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp 325 330 335 Pro GlnTrp Lys Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu 340 345 350 ValCys Ser Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp 355 360 365Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val 370 375380 Ser His Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly 385390 395 400 Leu Ala Leu 7 323 PRT Homo sapiens 7 Ser Asn Glu Leu His GlnVal Pro Ser Asn Cys Asp Cys Leu Asn Gly 1 5 10 15 Gly Thr Cys Val SerAsn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn 20 25 30 Cys Pro Lys Lys PheGly Gly Gln His Cys Glu Ile Asp Lys Ser Lys 35 40 45 Pro Ser Ser Pro ProGlu Glu Leu Lys Phe Gln Cys Gly Gln Lys Thr 50 55 60 Leu Arg Pro Arg PheLys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu 65 70 75 80 Asn Gln Pro TrpPhe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser 85 90 95 Val Thr Tyr ValCys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile 100 105 110 Ser Ala ThrHis Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile 115 120 125 Val TyrLeu Gly Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met 130 135 140 LysPhe Glu Val Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp 145 150 155160 Thr Leu Ala His His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys 165170 175 Glu Gly Arg Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu180 185 190 Pro Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu IleThr 195 200 205 Gly Phe Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro GluGln Leu 210 215 220 Lys Met Thr Val Val Lys Leu Ile Ser His Arg Glu CysGln Gln Pro 225 230 235 240 His Tyr Tyr Gly Ser Glu Val Thr Thr Lys MetLeu Cys Ala Ala Asp 245 250 255 Pro Gln Trp Lys Thr Asp Ser Cys Gln GlyAsp Ser Gly Gly Pro Leu 260 265 270 Val Cys Ser Leu Gln Gly Arg Met ThrLeu Thr Gly Ile Val Ser Trp 275 280 285 Gly Arg Gly Cys Ala Leu Lys AspLys Pro Gly Val Tyr Thr Arg Val 290 295 300 Ser His Phe Leu Pro Trp IleArg Ser His Thr Lys Glu Glu Asn Gly 305 310 315 320 Leu Ala Leu 8 143PRT Homo sapiens 8 Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp CysLeu Asn Gly 1 5 10 15 Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn IleHis Trp Cys Asn 20 25 30 Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu IleAsp Lys Ser Lys 35 40 45 Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg GlyLys Ala Ser Thr 50 55 60 Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn SerAla Thr Val Leu 65 70 75 80 Gln Gln Thr Tyr His Ala His Arg Ser Asp AlaLeu Gln Leu Gly Leu 85 90 95 Gly Lys His Asn Tyr Cys Arg Asn Pro Asp AsnArg Arg Arg Pro Trp 100 105 110 Cys Tyr Val Gln Val Gly Leu Lys Pro LeuVal Gln Glu Cys Met Val 115 120 125 His Asp Cys Ala Asp Gly Lys Lys ProSer Ser Pro Pro Glu Glu 130 135 140 9 96 PRT Homo sapiens 9 Lys Thr CysTyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser 1 5 10 15 Thr AspThr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val 20 25 30 Leu GlnGln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly 35 40 45 Leu GlyLys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro 50 55 60 Trp CysTyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met 65 70 75 80 ValHis Asp Cys Ala Asp Gly Lys Lys Pro Ser Ser Pro Pro Glu Glu 85 90 95 10264 DNA Homo sapiens 10 aaaacctgct atgaggggaa tggtcacttt taccgaggaaaggccagcac tgacaccatg 60 ggccggccct gcctgccctg gaactctgcc actgtccttcagcaaacgta ccatgcccac 120 agatctgatg ctcttcagct gggcctgggg aaacataattactgcaggaa cccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagccgcttgtcca agagtgcatg 240 gtgcatgact gcgcagatgg aaaa 264 11 141 DNA Homosapiens 11 agcaatgaac ttcatcaagt tccatcgaac tgtgactgtc taaatggaggaacatgtgtg 60 tccaacaagt acttctccaa cattcactgg tgcaactgcc caaagaaattcggagggcag 120 cactgtgaaa tagataagtc a 141 12 1236 DNA Homo sapiens 12agcaatgaac ttcatcaagt tccatcgaac tgtgactgtc taaatggagg aacatgtgtg 60tccaacaagt acttctccaa cattcactgg tgcaactgcc caaagaaatt cggagggcag 120cactgtgaaa tagataagtc aaaaacctgc tatgagggga atggtcactt ttaccgagga 180aaggccagca ctgacaccat gggccggccc tgcctgccct ggaactctgc cactgtcctt 240cagcaaacgt accatgccca cagatctgat gctcttcagc tgggcctggg gaaacataat 300tactgcagga acccagacaa ccggaggcga ccctggtgct atgtgcaggt gggcctaaag 360ccgcttgtcc aagagtgcat ggtgcatgac tgcgcagatg gaaaaaagcc ctcctctcct 420ccagaagaat taaaatttca gtgtggccaa aagactctga ggccccgctt taagattatt 480gggggagaat tcaccaccat cgagaaccag ccctggtttg cggccatcta caggaggcac 540cgggggggct ctgtcaccta cgtgtgtgga ggcagcctca tcagcccttg ctgggtgatc 600agcgccacac actgcttcat tgattaccca aagaaggagg actacatcgt ctacctgggt 660cgctcaaggc ttaactccaa cacgcaaggg gagatgaagt ttgaggtgga aaacctcatc 720ctacacaagg actacagcgc tgacacgctt gctcaccaca acgacattgc cttgctgaag 780atccgttcca aggagggcag gtgtgcgcag ccatcccgga ctatacagac catctgcctg 840ccctcgatgt ataacgatcc ccagtttggc acaagctgtg agatcactgg ctttggaaaa 900gagaattcta ccgactatct ctatccggag cagctgaaaa tgactgttgt gaagctgatt 960tcccaccggg agtgtcagca gccccactac tacggctctg aagtcaccac caaaatgcta 1020tgtgctgctg acccccaatg gaaaacagat tcctgccagg gagactcagg gggacccctc 1080gtctgttccc tccaaggccg catgactttg actggaattg tgagctgggg ccgtggatgt 1140gccctgaagg acaagccagg cgtctacacg agagtctcac acttcttacc ctggatccgc 1200agtcacacca aggaagagaa tggcctggcc ctctga 1236 13 405 DNA Homo sapiens 13agcaatgaac ttcatcaagt tccatcgaac tgtgactgtc taaatggagg aacatgtgtg 60tccaacaagt acttctccaa cattcactgg tgcaactgcc caaagaaatt cggagggcag 120cactgtgaaa tagataagtc aaaaacctgc tatgagggga atggtcactt ttaccgagga 180aaggccagca ctgacaccat gggccggccc tgcctgccct ggaactctgc cactgtcctt 240cagcaaacgt accatgccca cagatctgat gctcttcagc tgggcctggg gaaacataat 300tactgcagga acccagacaa ccggaggcga ccctggtgct atgtgcaggt gggcctaaag 360ccgcttgtcc aagagtgcat ggtgcatgac tgcgcagatg gaaaa 405 14 831 DNA Homosapiens 14 aagccctcct ctcctccaga agaattaaaa tttcagtgtg gccaaaagactctgaggccc 60 cgctttaaga ttattggggg agaattcacc accatcgaga accagccctggtttgcggcc 120 atctacagga ggcaccgggg gggctctgtc acctacgtgt gtggaggcagcctcatcagc 180 ccttgctggg tgatcagcgc cacacactgc ttcattgatt acccaaagaaggaggactac 240 atcgtctacc tgggtcgctc aaggcttaac tccaacacgc aaggggagatgaagtttgag 300 gtggaaaacc tcatcctaca caaggactac agcgctgaca cgcttgctcaccacaacgac 360 attgccttgc tgaagatccg ttccaaggag ggcaggtgtg cgcagccatcccggactata 420 cagaccatct gcctgccctc gatgtataac gatccccagt ttggcacaagctgtgagatc 480 actggctttg gaaaagagaa ttctaccgac tatctctatc cggagcagctgaaaatgact 540 gttgtgaagc tgatttccca ccgggagtgt cagcagcccc actactacggctctgaagtc 600 accaccaaaa tgctatgtgc tgctgacccc caatggaaaa cagattcctgccagggagac 660 tcagggggac ccctcgtctg ttccctccaa ggccgcatga ctttgactggaattgtgagc 720 tggggccgtg gatgtgccct gaaggacaag ccaggcgtct acacgagagtctcacacttc 780 ttaccctgga tccgcagtca caccaaggaa gagaatggcc tggccctctg a831 15 1212 DNA Homo sapiens 15 agcaatgaac ttcatcaagt tccatcgaactgtgactgtc taaatggagg aacatgtgtg 60 tccaacaagt acttctccaa cattcactggtgcaactgcc caaagaaatt cggagggcag 120 cactgtgaaa tagataagtc aaaaacctgctatgagggga atggtcactt ttaccgagga 180 aaggccagca ctgacaccat gggccggccctgcctgccct ggaactctgc cactgtcctt 240 cagcaaacgt accatgccca cagatctgatgctcttcagc tgggcctggg gaaacataat 300 tactgcagga acccagacaa ccggaggcgaccctggtgct atgtgcaggt gggcctaaag 360 ccgcttgtcc aagagtgcat ggtgcatgactgcgcagatg gaaaattaaa atttcagtgt 420 ggccaaaaga ctctgaggcc ccgctttaagattattgggg gagaattcac caccatcgag 480 aaccagccct ggtttgcggc catctacaggaggcaccggg ggggctctgt cacctacgtg 540 tgtggaggca gcctcatcag cccttgctgggtgatcagcg ccacacactg cttcattgat 600 tacccaaaga aggaggacta catcgtctacctgggtcgct caaggcttaa ctccaacacg 660 caaggggaga tgaagtttga ggtggaaaacctcatcctac acaaggacta cagcgctgac 720 acgcttgctc accacaacga cattgccttgctgaagatcc gttccaagga gggcaggtgt 780 gcgcagccat cccggactat acagaccatctgcctgccct cgatgtataa cgatccccag 840 tttggcacaa gctgtgagat cactggctttggaaaagaga attctaccga ctatctctat 900 ccggagcagc tgaaaatgac tgttgtgaagctgatttccc accgggagtg tcagcagccc 960 cactactacg gctctgaagt caccaccaaaatgctatgtg ctgctgaccc ccaatggaaa 1020 acagattcct gccagggaga ctcagggggacccctcgtct gttccctcca aggccgcatg 1080 actttgactg gaattgtgag ctggggccgtggatgtgccc tgaaggacaa gccaggcgtc 1140 tacacgagag tctcacactt cttaccctggatccgcagtc acaccaagga agagaatggc 1200 ctggccctct ga 1212 16 972 DNA Homosapiens 16 agcaatgaac ttcatcaagt tccatcgaac tgtgactgtc taaatggaggaacatgtgtg 60 tccaacaagt acttctccaa cattcactgg tgcaactgcc caaagaaattcggagggcag 120 cactgtgaaa tagataagtc aaagccctcc tctcctccag aagaattaaaatttcagtgt 180 ggccaaaaga ctctgaggcc ccgctttaag attattgggg gagaattcaccaccatcgag 240 aaccagccct ggtttgcggc catctacagg aggcaccggg ggggctctgtcacctacgtg 300 tgtggaggca gcctcatcag cccttgctgg gtgatcagcg ccacacactgcttcattgat 360 tacccaaaga aggaggacta catcgtctac ctgggtcgct caaggcttaactccaacacg 420 caaggggaga tgaagtttga ggtggaaaac ctcatcctac acaaggactacagcgctgac 480 acgcttgctc accacaacga cattgccttg ctgaagatcc gttccaaggagggcaggtgt 540 gcgcagccat cccggactat acagaccatc tgcctgccct cgatgtataacgatccccag 600 tttggcacaa gctgtgagat cactggcttt ggaaaagaga attctaccgactatctctat 660 ccggagcagc tgaaaatgac tgttgtgaag ctgatttccc accgggagtgtcagcagccc 720 cactactacg gctctgaagt caccaccaaa atgctatgtg ctgctgacccccaatggaaa 780 acagattcct gccagggaga ctcaggggga cccctcgtct gttccctccaaggccgcatg 840 actttgactg gaattgtgag ctggggccgt ggatgtgccc tgaaggacaagccaggcgtc 900 tacacgagag tctcacactt cttaccctgg atccgcagtc acaccaaggaagagaatggc 960 ctggccctct ga 972 17 429 DNA Homo sapiens 17 agcaatgaacttcatcaagt tccatcgaac tgtgactgtc taaatggagg aacatgtgtg 60 tccaacaagtacttctccaa cattcactgg tgcaactgcc caaagaaatt cggagggcag 120 cactgtgaaatagataagtc aaaaacctgc tatgagggga atggtcactt ttaccgagga 180 aaggccagcactgacaccat gggccggccc tgcctgccct ggaactctgc cactgtcctt 240 cagcaaacgtaccatgccca cagatctgat gctcttcagc tgggcctggg gaaacataat 300 tactgcaggaacccagacaa ccggaggcga ccctggtgct atgtgcaggt gggcctaaag 360 ccgcttgtccaagagtgcat ggtgcatgac tgcgcagatg gaaaaaagcc ctcctctcct 420 ccagaagaa 42918 288 DNA Homo sapiens 18 aaaacctgct atgaggggaa tggtcacttt taccgaggaaaggccagcac tgacaccatg 60 ggccggccct gcctgccctg gaactctgcc actgtccttcagcaaacgta ccatgcccac 120 agatctgatg ctcttcagct gggcctgggg aaacataattactgcaggaa cccagacaac 180 cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagccgcttgtcca agagtgcatg 240 gtgcatgact gcgcagatgg aaaaaagccc tcctctcctccagaagaa 288

What is claimed is:
 1. A composition comprising an isolatedurokinase-type plasminogen activator (uPA) kringle in an amounteffective to increase the contractility of a mammalian muscle orendothelial cell or tissue, wherein said uPA kringle consists of apolypeptide having the amino acid sequence corresponding to SEQ ID NO:1.2. The composition of claim 1, further comprising uPA connectingpeptide.
 3. A composition comprising a polypeptide, said polypeptide(low molecular weight domain of uPA) comprising the connecting peptideand protease domains of uPA in an amount effective to inhibit thecontractility of a mammalian muscle cell or tissue, wherein saidpolypeptide consists of a polypeptide having the amino acid sequencecorresponding to SEQ ID NO:5.
 4. The composition of claim 1, whereinsaid cell is in a mammal.
 5. The composition of claim 1, wherein saidmuscle cell is a smooth muscle cell and wherein said muscle tissue is asmooth muscle tissue.
 6. The composition of claim 1, further comprisingan inducing compound in an amount effective to mediate the contractionof a mammalian muscle cell or tissue, wherein said inducing compound isphenylepherine.
 7. The composition of claim 2, comprising an isolatedamino terminal fragment (ATE) of uPA, wherein said ATF consists of apolypeptide having the amino acid sequence corresponding to SEQ ID NO:4.8. The composition of claim 2, wherein said cell or tissue is a vascularsmooth muscle cell.
 9. The composition of claim 2, comprising thedeletion mutant polypeptide scuPA^((Δ136-143)) in an a mount effectiveto increase the contractility of a mammalian muscle cell or tissue,wherein said scuPA^((Δ136-143)) consists of a polypeptide having theamino acid sequence corresponding to SEQ ID NO:6.
 10. The composition ofclaim 2, wherein said composition further comprises a polypeptide, saidpolypeptide comprising the amino terminal fragment (ATF) and theconnecting peptide of uPA, wherein said polypeptide consists of apolypeptide having the amino acid sequence corresponding to SEQ ID NO:8.11. The composition of claim 2, wherein said composition furthercomprises a polypeptide, said polypeptide comprising the uPA kringle andthe connecting peptide, wherein said polypeptide consists of apolypeptide having the amino acid sequence corresponding to SEQ ID NO:9.12. The composition of claim 1, wherein said composition is in the formof a pharmaceutical composition.
 13. A composition comprising apolypeptide, said polypeptide consisting of an amino acid sequence ofcorresponding to SEQ ID NO:8, wherein said polypeptide is present in anamount effective to increase the contractility of a mammalian muscle orendothelial cell or tissue.
 14. A kit for treating a disease orcondition in a mammal, wherein a symptom of said disease or conditioncomprises abnormal muscle cell or tissue contractility, said kitcomprising a) a composition comprising a polypeptide, said polypeptidecomprising a uPA kringle in an amount effective to increase thecontractility of a mammalian muscle or endothelial cell or tissue; andb) an instructional material.
 15. The kit of claim 14, furthercomprising a sterile solvent suitable for dissolving or suspending saidcomposition prior to administering said composition to said mammal.